Metalloproteinases

ABSTRACT

The present invention relates to novel metalloproteinase-like proteins. In particular, isolated nucleic acid molecules are provided encoding the human TACE-like and matrilysin-like proteins. TACE-like and matrilysin-like polypeptides are also provided as are vectors, host cells and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists and antagonists of TACE-like and matrilysin-like activity. Also provided are diagnostic methods for detecting cancer and therapeutic methods for cancer and other disorders characterized by an over or under production of these metalloproteinases.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of and claims priority under 35 U.S.C.§ 120 to U.S. application Ser. No. 09/372,154, filed Aug. 11, 1999, thatissued as U.S. Pat. No. 6,312,937 on Nov. 6, 2001, which is a Divisionalof, claiming priority under 35 U.S.C. § 120 to, U.S. application Ser.No. 09/009,156, filed Jan. 20, 1998, that issued as U.S. Pat. No.6,046,031 on Apr. 4, 2000, which claims priority under 35 U.S.C. §119(e)to U.S. Provisional Applications Serial Nos: 60/034,205, filed Jan. 21,1997, 60/049,607, filed Jun. 13, 1997 and 60/054,541, filed Aug. 1,1997, all of which are incorporated herein by reference in theirentireties.

FIELD OF INVENTION

The present invention relates to novel metalloproteinases. Morespecifically, isolated nucleic acid molecules are provided encoding: (1)a human TNF-alpha converting enzyme (“TACE”)-like protein, and (2) amatrilysin-like protein. TACE-like and matrilysin-like polypeptides arealso provided, as are vectors, host cells, and recombinant methods forproducing the same. The invention further relates to screening methodsfor identifying agonists and antagonists of the activity of these twoenzymes. Also provided are diagnostic and therapeutic methods fordetecting and treating diseases or disorders that involve these enzymes.

BACKGROUND OF THE INVENTION

Tumor necrosis factor-alpha (TNF-α) is a potent cytokine, secretedprimarily by activated monocytes and macrophages, that contributes to avariety of inflammatory disease states and is broadly involved inimmunomodulation. TNF-alpha is processed from an immature,membrane-bound form to a mature, secreted form by a metalloproteinasecalled TNF-alpha converting enzyme, or “TACE.” See, R. A. Black et al.,Nature 385:729-733 (February 1997); M. L. Moss et al., Nature385:733-736 (February 1997); M. L. Moss et al., J. Neuroimmunol.72:127-129 (February 1997). Inhibitors of the enzyme TACE blocksecretion of TNF-alpha.

TACE is a new member of a protein family called “ADAMs” (proteins whichcontain A Disintegrin And Metalloprotease domain; also calledadamalysins). See, Wolfsberg et al., Dev. Biol. 169:378-383 (1995).

The TACE/ADAM family is composed of membrane proteins with structuralhomology to the snake venom metalloproteases and disintegrins. Snakevenom disintegrins are a family of anticoagulant peptides with a highcysteine content. A new member of TACE/ADAM in Drosophila, called thekuzbanian gene (“KUZ”), was found to be involved in Drosophilaneurogenesis (Rooke, J. et al., Science 273:1227-1231 (August 1996)).

Approximately 11-13 ADAM genes have now been identified, includingfertilin alpha and beta (involved in the integrin mediated binding andfusion of egg and sperm; previously known as PH-30 alpha and beta),epididymal apical protein I, cyritestin, MDC (a candidate for tumorsuppressor in human breast cancer), meltrin- (mediates fusion ofmyoblast fusion in the process of myotube formation), MS2 (a macrophagesurface antigen), and metargidin. Typical ADAMs are cell surfaceproteins which consist of pro-, metalloprotease-like, disintegrin-like,cysteine-rich, epidermal growth factor-like repeat, transmembrane andcytoplasmic domains.

A new ADAM family gene, named ADAMTS-1, containing a disintegrin andmetalloproteinase domain with thrombospondin (TSP) motifs, has now beenshown to be closely associated with various inflammatory processes, aswell as development of cancer cachexia. Kuno, K. et al., J. Biol. Chem.272:556-562 (1997).

The disintegrin domain of ADAM family proteins functions in theprevention of integrin-mediated cell to cell and cell to matrixinteractions, such as platelet aggregation, adhesion, and migration oftumor cells or neutrophils, and angiogenesis. Previously describeddisintegrins, such as contortrostatin (Trikha et al., Cancer Research54:4993-4998 (1994) have been used to inhibit human metastatic melanoma(M24 cells) cell adhesion to type I collagen, vitronectin, andfibronection, but not laminin. Further, contortrostatin inhibits lungcolonization of M24 cells in a murine metastasis model.

The matrix metalloproteinases (MMPs) compose a family of structurallysimilar zinc-dependent enzymes that degrade all of the major componentsof the extracellular matrix. MMPs include the collagenases, gelatinasesA and B, the stromelysins, matrilysin, metalloelastase, and themembrane-type matrix metalloproteinases. Over-expression and activationof MMPs have been linked with a range of diseases, such as arthritis,cancer, and multiple sclerosis. Regarding cancer, although MMPsclassically have been implicated in basement membrane destructionassociated with late-stage tumor cell invasion and metastasis, one MMPmember, matrilysin, has recently been shown to be expressed in earlystage human colorectal tumors. (C. L. Wilson et al., Proc. Natl. Acad.Sci. USA 94:1402-1407 (February 1997)).

Clearly, members of the TACE/ADAM family of proteins have a highpotential for becoming valuable therapeutically and diagnostically. ADAMproteins, peptides derived from the sequence of ADAM proteins, and ADAMprotein antagonists may become desirable components of molecular methodsof assisting or preventing fertilization. Furthermore, specificTACE/ADAM proteins or derivatives may be useful in the detection andprevention of muscle disorders. TACE-like proteins also have an excitingpotential potential in the treatment of inflammation, thrombosis,cancer, and cancer metastasis. TACE-like factors, or antagoniststhereof, may also become useful agents in promoting macrophage or T-celladhesion to matrices or cells' access to bound cytokines and otherregulatory molecules.

Inhibitors of members of the matrix metalloproteinase (MMP) family (suchas matrilysin) have been studied in the treatment or prophylaxis ofcancer, cancer metastasis, as well as in the treatment of arthritis,corneal ulcers, pleural effusion, and multiple sclerosis. For a reviewon recent advances in matrix metalloproteinase research, see, Beckett,R. P. et al., DDT 1:16-26 (January 1996).

Clearly, there is a need in the art for novel TACE-like andmatrilysin-like molecules, exhibiting sequence relatedness to knownmetalloproteinases with recognized therapeutic and diagnosticusefulness.

SUMMARY OF THE INVENTION

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding the TACE-like polypeptide havingthe amino acid sequence shown in SEQ ID NO:2 or the amino acid sequenceencoded by the cDNA clone deposited as ATCC Deposit Number 209042 on May15, 1997. The present invention also provides isolated nucleic acidmolecules comprising a polynucleotide encoding the matrilysin-likepolypeptide having the amino acid sequence shown in SEQ ID NO: 5 or theamino acid sequence encoded by the cDNA clone deposited as ATCC DepositNumber 209055 on May 16, 1997.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofTACE-like or matrilysin-like polypeptides or peptides by recombinanttechniques.

The invention further provides isolated TACE-like or matrilysin-likepolypeptides having an amino acid sequence encoded by a polynucleotidedescribed herein.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a cellular response inducedby the TACE-like or matrilysin-like protein, which involves contactingcells which express the TACE-like or matrilysin-like protein with thecandidate compound, assaying a cellular response, and comparing thecellular response to a standard cellular response, the standard beingassayed when contact is made in absence of the candidate compound;whereby, an increased cellular response over the standard indicates thatthe compound is an agonist and a decreased cellular response over thestandard indicates that the compound is an antagonist.

The invention provides a diagnostic method useful during diagnosis ofcancer.

An additional aspect of the invention is related to a method fortreating an individual in need of an increased level of TACE-likeactivity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of an isolatedTACE-like polypeptide of the invention or an agonist thereof.

An additional aspect of the invention is related to a method fortreating an individual in need of a decreased level of TACE-likeactivity in the body comprising administering to such an individual acomposition comprising a therapeutically effective amount of a TACE-likepolypeptide antagonist.

A still further aspect of the invention is related to a method fortreating an individual in need of a decreased level of matrilysin-likeactivity in the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of amatrilysin-like antagonist.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the nucleotide (SEQ ID NO:1) and deduced amino acid (SEQ IDNO:2) sequences of the TACE-like protein of the invention. The proteinhas a leader sequence of about 15 amino acid residues (first underlinedportion) and a deduced molecular weight of about 20.9 kDa. It is furtherpredicted that amino acid residues from about 169 to about 182 (secondunderlined portion) constitute the transmembrane domain.

FIG. 2 shows the regions of similarity between the amino acid sequencesof the TACE-like polypeptide and tMDC II (SEQ ID NO:3).

FIG. 3 shows an analysis of the TACE-like amino acid sequence. Alpha,beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues about 13 to about 22, about 31 to about 46, 55 toabout 103, 123 to about 151, and 159 to about 173 in FIG. 1 correspondto the shown highly antigenic regions of the TACE-like protein.

FIGS. 4A and B show the nucleotide (SEQ ID NO:4) and deduced amino acid(SEQ ID NO:5) sequences of the matrilysin-like protein of the invention.The protein has a leader sequence of about 22 amino acid residues(underlined) and a deduced molecular weight of about 29.7 kDa.

FIG. 5 shows the regions of similarity between the amino acid sequencesof the matrilysin-like protein and human matrilysin (SEQ ID NO:6).

FIG. 6 shows an analysis of the matrilysin-like amino acid sequence.Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity;amphipathic regions; flexible regions; antigenic index and surfaceprobability are shown. In the “Antigenic Index—Jameson-Wolf” graph,amino acid residues about 24 to about 71, about 81 to about 125, 141 toabout 167, 178 to about 202, and 212 to about 260 in FIG. 1 correspondto the shown highly antigenic regions of the matrilysin-like protein.

FIG. 7 shows sequence HTEBO72R (SEQ ID NO: 7); HETAG43R (SEQ ID NO: 9)and HETAF71R (SEQ ID NO: 8).

FIG. 8 shows the nucleotide sequence (SEQ ID NO:20) derived from HETAF71as it appeared in U.S. Provisional Patent Application Serial No.60/049,607.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides isolated nucleic acid moleculescomprising a polynucleotide encoding a TACE-like or matrilysin-likepolypeptide having the amino acid sequence shown in SEQ ID NO:2 or SEQID NO:5, respectively, which was determined by sequencing cloned cDNA.

The TACE-like protein of the present invention shares sequence homologywith tMDC II (FIG. 2) (SEQ ID NO:3). The nucleotide sequence shown inSEQ ID NO:1 was obtained by sequencing a cDNA clone (“HTEJQ70”), whichwas deposited on May 15, 1997 at the American Type Culture Collection,10801 University Blvd., Manassas, Va. 20110, and given accession number209042. The deposited clone is inserted in the pBluescript SK(−) plasmid(Stratagene, LaJolla, Calif.).

The matrilysin-like protein of the present invention shares sequencehomology with human matrilysin (FIG. 2) (SEQ ID NO:6). The nucleotidesequence shown in SEQ ID NO:4 was obtained by sequencing a cDNA clone(“HETBW05”), which was deposited on May 16, 1997 at the American TypeCulture Collection, 10801 University Blvd., Manassas, Va. 20110, andgiven accession number 209055. The deposited clone is inserted in thepBluescript SK(−) plasmid (Stratagene, LaJolla, Calif.). A related clonehas also been discovered the polynucleotide sequence of which is shownin FIG. 8. This clone, HETAF71, was also deposited with the ATCC, alongwith other cDNA clones as a mixture, on Jun. 5, 1997 and given accessionnumber 209090. These clones are believed to be derived from the samegene and are further believed to encode the same polypeptide.Discrepancies between the sequence shown in FIG. 4A and B (SEQ ID NO:4)and the sequence shown in FIG. 8 (SEQ ID NO:20) are believed to be dueto errors in the DNA sequencing process, as is described below. In caseof conflict, such discrepancies should be resolved in favor of SEQ IDNO:4 and clone HETBW05. The remaining description of the invention as itpertains to matrilysin-like protein references HETBW05 (SEQ ID NO:4) andthe polypeptide encoded thereby, however, it is the intention of theapplicants that HETAF71 (SEQ ID NO:8) and the protein encoded therebycould equally be substituted in place of HETBW05. Accordingly, it willbe appreciated that applicants intend to encompass HETAF71 within theinvention in all respects as it pertains to HETBW05.

Nucleic Acid Molecules

Unless otherwise indicated, all nucleotide sequences determined bysequencing a DNA molecule herein were determined using an automated DNAsequencer (such as the Model 373 from Applied Biosystems, Inc.), and allamino acid sequences of polypeptides encoded by DNA molecules determinedherein were predicted by translation of a DNA sequence determined asabove. Therefore, as is known in the art for any DNA sequence determinedby this automated approach, any nucleotide sequence determined hereinmay contain some errors. Nucleotide sequences determined by automationare typically at least about 90% identical, more typically at leastabout 95% to at least about 99.9% identical to the actual nucleotidesequence of the sequenced DNA molecule. The actual sequence can be moreprecisely determined by other approaches including manual DNA sequencingmethods well known in the art. As is also known in the art, a singleinsertion or deletion in a determined nucleotide sequence compared tothe actual sequence will cause a frame shift in translation of thenucleotide sequence such that the predicted amino acid sequence encodedby a determined nucleotide sequence will be completely different fromthe amino acid sequence actually encoded by the sequenced DNA molecule,beginning at the point of such an insertion or deletion.

Using the information provided herein, such as the nucleotide sequencein SEQ ID NO: 1 or SEQ ID NO:4, a nucleic acid molecule of the presentinvention encoding a TACE-like or matrilysin-like polypeptide may beobtained using standard cloning and screening procedures, such as thosefor cloning cDNAs using mRNA as starting material.

Illustrative of the invention, the nucleic acid molecule described inSEQ ID NO:1 was discovered in a cDNA library derived from human testes.The gene was also identified in cDNA libraries from amniotic cells(primary culture) and keratinocytes The determined nucleotide sequenceof the TACE-like cDNA of SEQ ID NO:1 contains an open reading frameencoding a protein of about 182 amino acid residues, a predicted leadersequence of about 15 amino acid residues, and a deduced molecular weightof about 20.9 kDa. The TACE-like protein shown in SEQ ID NO:2 is about44% identical and about 62% similar to tMDC II (FIG. 2).

Also illustrative of the invention, the nucleic acid molecule describedin SEQ ID NO:4 was discovered in a cDNA library derived from humanumbilical vein endothelial cells. The gene was also identified in cDNAlibraries from the following tissues: primarily human endometrial tumor;but also in human colon, CD34+ cells, human B-cell lymphoma, andendothelial induced human microvascular endothelial cells. Thedetermined nucleotide sequence of the matrilysin-like cDNA of SEQ IDNO:4 contains an open reading frame encoding a protein of about 261amino acid residues, a predicted leader sequence of about 22 amino acidresidues, and a deduced molecular weight of about 29.7 kDa. Thematrilysin-like protein shown in SEQ ID NO:5 is about 39% identical andabout 59% similar to human matrilysin (FIG. 5).

The present invention also provides the mature form(s) of the TACE-likeand matrilysin-like proteins of the present invention. According to thesignal hypothesis, proteins secreted by mammalian cells have a signal orsecretory leader sequence which is cleaved from the mature protein onceexport of the growing protein chain across the rough endoplasmicreticulum has been initiated. Most mammalian cells and even insect cellscleave secreted proteins with the same specificity. However, in somecases, cleavage of a secreted protein is not entirely uniform, whichresults in two or more mature species on the protein. Further, it haslong been known that the cleavage specificity of a secreted protein isultimately determined by the primary structure of the complete protein,that is, it is inherent in the amino acid sequence of the polypeptide.

Therefore, the present invention provides a nucleotide sequence encodingthe mature TACE-like polypeptide having the amino acid sequence encodedby the cDNA clone contained in the host identified as ATCC Deposit No.209042 and as shown in SEQ ID NO:2. By the mature TACE-like proteinhaving the amino acid sequence encoded by the cDNA clone contained inthe host identified as ATCC Deposit 209042 is meant the mature form(s)of the TACE-like protein produced by expression in a mammalian cell(e.g., COS cells, as described below) of the complete open reading frameencoded by the human DNA sequence of the clone contained in the vectorin the deposited host. As indicated below, the mature TACE-like proteinhaving the amino acid sequence encoded by the cDNA clone contained inATCC Deposit No.209042 may or may not differ from the predicted “mature”TACE-like protein shown in SEQ ID NO:2 (amino acids from about 16 toabout 182) depending on the accuracy of the predicted cleavage sitebased on computer analysis.

The present invention also provides a nucleotide sequence encoding themature matrilysin-like polypeptide having the amino acid sequenceencoded by the cDNA clone contained in the host identified as ATCCDeposit No.209055 and as shown in SEQ ID NO:5. By the maturematrilysin-like protein having the amino acid sequence encoded by thecDNA clone contained in the host identified as ATCC Deposit 209055 ismeant the mature form(s) of the matrilysin-like protein produced byexpression in a mammalian cell (e.g., COS cells, as described below) ofthe complete open reading frame encoded by the human DNA sequence of theclone contained in the vector in the deposited host. As indicated below,the mature matrilysin-like protein having the amino acid sequenceencoded by the cDNA clone contained in ATCC Deposit No.209055 may or maynot differ from the predicted “mature” matrilysin-like protein shown inSEQ ID NO:5 (amino acids from about 23 to about 261) depending on theaccuracy of the predicted cleavage site based on computer analysis.

Methods for predicting whether a protein has a secretory leader as wellas the cleavage point for that leader sequence are available. Forinstance, the methods of McGeoch (Virus Res. 3:271-286 (1985)) and vonHeinje (Nucleic Acids Res. 14:4683-4690 (1986)) can be used. Theaccuracy of predicting the cleavage points of known mammalian secretoryproteins for each of these methods is in the range of 75-80%. vonHeinje, supra. However, the two methods do not always produce the samepredicted cleavage point(s) for a given protein.

In the present case, the predicted amino acid sequence of the completeTACE-like and matrilysin-like polypeptides of the present invention wereanalyzed by a computer program (“PSORT”) (K. Nakai and M. Kanehisa,Genomics 14:897-911 (1992)), which is an expert system for predictingthe cellular location of a protein based on the amino acid sequence. Aspart of this computational prediction of localization, the methods ofMcGeoch and von Heinje are incorporated. The analysis by the PSORTprogram predicted the cleavage site between amino acids −1 and 1 in SEQID NOS:2 and 5. Thereafter, the complete amino acid sequences werefurther analyzed by visual inspection, applying a simple form of the(−1,−3) rule of von Heinje. von Heinje, supra.

Thus, the leader sequence for the TACE-like protein is predicted toconsist of amino acid residues from about 1 to about 15 in SEQ ID NO:2,while the mature TACE-like protein is predicted to consist of residuesfrom about 16 to about 182.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors, as well as the variability of cleavage sites forleaders in different known proteins, the predicted TACE-like polypeptideencoded by the deposited cDNA comprises about 182 amino acids, but maybe anywhere in the range of about 172 to about 192 amino acids; and thepredicted leader sequence of this protein is about 15 amino acids, butmay be anywhere in the range of about 5 to about 25 amino acids.

The leader sequence for the matrilysin-like protein is predicted toconsist of amino acid residues from about 1 to about 22 in SEQ ID NO:5,while the mature matrilysin-like protein is predicted to consist ofresidues from about 23 to about 261.

As one of ordinary skill would appreciate, due to the possibilities ofsequencing errors, as well as the variability of cleavage sites forleaders in different known proteins, the predicted matrilysin-likepolypeptide encoded by the deposited cDNA comprises about 261 aminoacids, but may be anywhere in the range of about 251 to about 271 aminoacids; and the predicted leader sequence of this protein is about 22amino acids, but may be anywhere in the range of about 12 to about 32amino acids.

As indicated, nucleic acid molecules of the present invention may be inthe form of RNA, such as mRNA, or in the form of DNA, including, forinstance, cDNA and genomic DNA obtained by cloning or producedsynthetically. The DNA may be double-stranded or single-stranded.Single-stranded DNA or RNA may be the coding strand, also known as thesense strand, or it may be the non-coding strand, also referred to asthe anti-sense strand.

By “isolated” nucleic acid molecule(s) is intended a nucleic acidmolecule, DNA or RNA, which has been removed from its native environmentFor example, recombinant DNA molecules contained in a vector areconsidered isolated for the purposes of the present invention. Furtherexamples of isolated DNA molecules include recombinant DNA moleculesmaintained in heterologous host cells or purified (partially orsubstantially) DNA molecules in solution. Isolated RNA molecules includein vivo or in vitro RNA transcripts of the DNA molecules of the presentinvention. Isolated nucleic acid molecules according to the presentinvention further include such molecules produced synthetically.

Isolated nucleic acid molecules of the present invention include DNAmolecules comprising an open reading frame (ORF) shown in SEQ ID NO:1 orSEQ ID NO:4; DNA molecules comprising the coding sequence for the matureTACE-like or matrilysin-like protein; and DNA molecules which comprise asequence substantially different from those described above but which,due to the degeneracy of the genetic code, still encode the TACE-like ormatrilysin-like protein protein. Of course, the genetic code is wellknown in the art. Thus, it would be routine for one skilled in the artto generate such degenerate variants.

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:1 whichhave been determined from the following related cDNA clone: HTEBO72R(SEQ ID NO:7).

In addition, the invention provides nucleic acid molecules havingnucleotide sequences related to extensive portions of SEQ ID NO:4, whichhave been determined from the following related cDNA clones: HETAF71R(SEQ ID NO:8) and HETAG43R (SEQ ID NO:9).

In another aspect, the invention provides isolated nucleic acidmolecules encoding the TACE-like polypeptide having an amino acidsequence as encoded by the cDNA clone contained in the plasmid depositedas ATCC Deposit No. 209042 on May 15, 1997. In a further embodiment,nucleic acid molecules are provided encoding the mature TACE-likepolypeptide or the full-length TACE-like polypeptide lacking theN-terminal methionine. The invention also provides an isolated nucleicacid molecule having the nucleotide sequence shown in SEQ ID NO:1 or thenucleotide sequence of the TACE-like cDNA contained in theabove-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences.

The invention also provides isolated nucleic acid molecules encoding thematrilysin-like polypeptide having an amino acid sequence as encoded bythe cDNA clone contained in the plasmid deposited as ATCC Deposit No.209055 on May 16, 1997. In a further embodiment, nucleic acid moleculesare provided encoding the mature matrilysin-like polypeptide or thefull-length matrilysin-like polypeptide lacking the N-terminalmethionine. The invention also provides an isolated nucleic acidmolecule having the nucleotide sequence shown in SEQ ID NO:4 or thenucleotide sequence of the matrilysin-like cDNA contained in theabove-described deposited clone, or a nucleic acid molecule having asequence complementary to one of the above sequences.

Such isolated molecules, discussed supra, particularly DNA molecules,are useful as probes for gene mapping, by in situ hybridization withchromosomes, and for detecting expression of the TACE-like ormatrilysin-like gene in human tissue, for instance, by Northern blotanalysis.

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having the nucleotide sequence of the depositedcDNA or the nucleotide sequence shown in SEQ ID NOS: 1 or 4 is intendedfragments at least about 15 nt, and more preferably at least about 20nt, still more preferably at least about 30 nt, and even morepreferably, at least about 40 nt in length which are useful asdiagnostic probes and primers as discussed herein. Of course, largerfragments 50-500 nt in length (TACE-like) and 50-700 nt in length(matrilysin-like) are also useful according to the present invention asare fragments corresponding to most, if not all, of the nucleotidesequence of the deposited cDNA or as shown in SEQ ID NOS:1 or 4. By afragment at least 20 nt in length, for example, is intended fragments,which include 20 or more contiguous bases from the nucleotide sequenceof the deposited cDNA or the nucleotide sequence as shown in SEQ IDNOS:1 or 4.

Preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding: a polypeptide comprising the TACE-liketransmembrane domain (predicted to constitute amino acid residues fromabout 169 to about 182 in SEQ ID NO:2) As above with the leadersequence, the amino acid residues constituting the TACE-liketransmembrane domain has been predicted by computer analysis. Thus, asone of ordinary skill would appreciate, the amino acid residuesconstituting this domains may vary slightly (e.g., by about 1 to about15 amino acid residues) depending on the criteria used to define eachdomain.

Preferred nucleic acid fragments of the present invention also includenucleic acid molecules encoding epitope-bearing portions of theTACE-like protein. In particular, such nucleic acid fragments of thepresent invention include nucleic acid molecules encoding: a polypeptidecomprising amino acid residues from about 13 to about 22 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 31 to about 46in SEQ ID NO:2; a polypeptide comprising amino acid residues from about55 to about 103 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 123 to about 151 in SEQ ID NO:2; and a polypeptidecomprising amino acid residues from about 159 to about 173 in SEQ IDNO:2. The inventors have determined that the above polypeptide fragmentsare antigenic regions of the TACE-like protein. Methods for determiningother such epitope-bearing portions of the TACE-like protein aredescribed in detail below.

Other preferred nucleic acid fragments of the present invention includenucleic acid molecules encoding epitope-bearing portions of thematrilysin-like protein. In particular, such nucleic acid fragments ofthe present invention include nucleic acid molecules encoding: apolypeptide comprising amino acid residues from about 24 to about 71 inSEQ ID NO:5; a polypeptide comprising amino acid residues from about 81to about 125 in SEQ ID NO:5; a polypeptide comprising amino acidresidues from about 141 to about 167 in SEQ ID NO:5; a polypeptidecomprising amino acid residues from about 173 to about 202 in SEQ IDNO:5; and a polypeptide comprising amino acid residues from about 212 toabout 260 in SEQ ID NO:5. The inventors have determined that the abovepolypeptide fragments are antigenic regions of the matrilysin-likeprotein. Methods for determining other such epitope-bearing portions ofthe matrilysin-like protein are described in detail below.

In another aspect, the invention provides an isolated nucleic acidmolecule comprising a polynucleotide which hybridizes under stringenthybridization conditions to a portion of the polynucleotide in a nucleicacid molecule of the invention described above, for instance, the cDNAclone contained in ATCC Deposit 209042 or ATCC Deposit 209055. By“stringent hybridization conditions” is intended overnight incubation at42 C. in a solution comprising: 50% formamide, 5×SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5×Denhardt'ssolution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmonsperm DNA, followed by washing the filters in 0.1×SSC at about 65 C.

By a polynucleotide which hybridizes to a “portion” of a polynucleotideis intended a polynucleotide (either DNA or RNA) hybridizing to at leastabout 15 nucleotides (nt), and more preferably at least about 20 nt,still more preferably at least about 30 nt, and even more preferablyabout 30-70 nt of the reference polynucleotide. These are useful asdiagnostic probes and primers as discussed above and in more detailbelow.

By a portion of a polynucleotide of “at least 20 nt in length,” forexample, is intended 20 or more contiguous nucleotides from thenucleotide sequence of the reference polynucleotide (e.g., the depositedcDNA or the nucleotide sequence as shown in SEQ ID NO:1 or SEQ ID NO:4).Of course, a polynucleotide which hybridizes only to a poly A sequence(such as the 3 terminal poly(A) tract of the TACE-like cDNA shown in SEQID NO:1 or the matrilysin-like cDNA shown in SEQ ID NO:4), or to acomplementary stretch of T (or U) resides, would not be included in apolynucleotide of the invention used to hybridize to a portion of anucleic acid of the invention, since such a polynucleotide wouldhybridize to any nucleic acid molecule containing a poly (A) stretch orthe complement thereof (e.g., practically any double-stranded cDNAclone). Particularly preferred regions for selecting such fragmentsinclude the coding regions shown in FIGS. 1 and 4A-B; i.e., nucleotides22 through 567 of SEQ ID NO:1 and nucleotides 46 through 827 of SEQ IDNO:4.

As indicated, nucleic acid molecules of the present invention whichencode a TACE-like or matrilysin-like polypeptide may include, but arenot limited to those encoding the amino acid sequence of the maturepolypeptide, by itself; the coding sequence for the mature polypeptideand additional sequences, such as those encoding the leader or secretorysequence, such as a pre-, or pro- or prepro- protein sequence; thecoding sequence of the mature polypeptide, with or without theaforementioned additional coding sequences, together with additional,non-coding sequences, including for example, but not limited to intronsand non-coding 5 and 3 sequences, such as the transcribed,non-translated sequences that play a role in transcription, mRNAprocessing, including splicing and polyadenylation signals, forexample—ribosome binding and stability of mRNA; an additional codingsequence which codes for additional amino acids, such as those whichprovide additional functionalities. Thus, the sequence encoding thepolypeptide may be fused to a marker sequence, such as a sequenceencoding a peptide which facilitates purification of the fusedpolypeptide. In certain preferred embodiments of this aspect of theinvention, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (Qiagen, Inc.), among others,many of which are commercially available. As described in Gentz et al.,Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. The “HA” tag is another peptide useful for purification whichcorresponds to an epitope derived from the influenza hemagglutininprotein, which has been described by Wilson et al., Cell 37:767-778(1984). As discussed below, other such fusion proteins include theTACE-like or matrilysin-like protein fused to Fc at the N- orC-terminus.

The present invention further relates to variants of the nucleic acidmolecules of the present invention, which encode portions, analogs orderivatives of the TACE-like or matrilysin-like protein. Variants mayoccur naturally, such as a natural allelic variant. By an “allelicvariant” is intended one of several alternate forms of a gene occupyinga given locus on a chromosome of an organism. Genes II, Lewin, B., ed.,John Wiley & Sons, New York (1985). Non-naturally occurring variants maybe produced using art-known mutagenesis techniques.

Such variants include those produced by nucleotide substitutions,deletions or additions, which may involve one or more nucleotides. Thevariants may be altered in coding regions, non-coding regions, or both.Alterations in the coding regions may produce conservative ornon-conservative amino acid substitutions, deletions or additions.Especially preferred among these are silent substitutions, additions anddeletions, which do not alter the properties and activities of theTACE-like or matrilysin-like protein or portions thereof. Alsoespecially preferred in this regard are conservative substitutions.

Further embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the polypeptidehaving the amino acid sequence in SEQ ID NO:2; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:2,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 16 to about 182 in SEQ ID NO:2; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No. 209042; (e) a nucleotidesequence encoding the mature TACE-like polypeptide having the amino acidsequence encoded by the cDNA clone contained in ATCC Deposit No.209042;(f) a nucleotide sequence encoding the TACE-like transmembrane domain;or (g) a nucleotide sequence complementary to any of the nucleotidesequences in (a), (b), (c), (d), (e), or (f).

Additional embodiments of the invention include isolated nucleic acidmolecules comprising a polynucleotide having a nucleotide sequence atleast 90% identical, and more preferably at least 95%, 96%, 97%, 98% or99% identical to (a) a nucleotide sequence encoding the polypeptidehaving the amino acid sequence in SEQ ID NO:5; (b) a nucleotide sequenceencoding the polypeptide having the amino acid sequence in SEQ ID NO:5,but lacking the N-terminal methionine; (c) a nucleotide sequenceencoding the polypeptide having the amino acid sequence at positionsfrom about 23 to about 261 in SEQ ID NO:5; (d) a nucleotide sequenceencoding the polypeptide having the amino acid sequence encoded by thecDNA clone contained in ATCC Deposit No.209055; (e) a nucleotidesequence encoding the mature matrilysin-like polypeptide having theamino acid sequence encoded by the cDNA clone contained in ATCC DepositNo.209055; or (f) a nucleotide sequence complementary to any of thenucleotide sequences in (a), (b), (c), (d), or (e).

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence encoding a TACE-likeor matrilysin-like polypeptide is intended that the nucleotide sequenceof the polynucleotide is identical to the reference sequence except thatthe polynucleotide sequence may include up to five point mutations pereach 100 nucleotides of the reference nucleotide sequence encoding theTACE-like or matrilysin-like polypeptide. In other words, to obtain apolynucleotide having a nucleotide sequence at least 95% identical to areference nucleotide sequence, up to 5% of the nucleotides in thereference sequence may be deleted or substituted with anothernucleotide, or a number of nucleotides up to 5% of the total nucleotidesin the reference sequence may be inserted into the reference sequence.These mutations of the reference sequence may occur at the 5′ or 3′terminal positions of the reference nucleotide sequence or anywherebetween those terminal positions, interspersed either individually amongnucleotides in the reference sequence or in one or more contiguousgroups within the reference sequence.

As a practical matter, whether any particular nucleic acid molecule isat least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, thenucleotide sequence shown in SEQ ID NO:1 or SEQ ID NO:4, or to thenucleotide sequence of the deposited cDNA clone can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). Bestfit uses the local homology algorithm of Smith andWaterman, Advances in Applied Mathematics 2: 482-489 (1981), to find thebest segment of homology between two sequences. When using Bestfit orany other sequence alignment program to determine whether a particularsequence is, for instance, 95% identical to a reference sequenceaccording to the present invention, the parameters are set, of course,such that the percentage of identity is calculated over the full lengthof the reference nucleotide sequence and that gaps in homology of up to5% of the total number of nucleotides in the reference sequence areallowed.

The present application is directed to nucleic acid molecules at least90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequenceshown in SEQ ID NO:1 or SEQ ID NO:4, or to the nucleic acid sequence ofthe deposited cDNA, irrespective of whether they encode a polypeptidehaving TACE-like or matrilysin-like activity. This is because even wherea particular nucleic acid molecule does not encode a polypeptide havingTACE-like or matrilysin-like activity, one of skill in the art wouldstill know how to use the nucleic acid molecule, for instance, as ahybridization probe or a polymerase chain reaction (PCR) primer. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having TACE-like or matrilysin-like activity include, interalia, (1) isolating the TACE-like or matrilysin-like gene or allelicvariants thereof in a cDNA library; (2) in situ hybridization (e.g.,“FISH”) to metaphase chromosomal spreads to provide precise chromosomallocation of the TACE-like or matrilysin-like genes, as described inVerma et al., Human Chromosomes: A Manual of Basic Techniques, PergamonPress, New York (1988); and Northern Blot analysis for detectingTACE-like or matrilysin-like mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least90%, 95%, 96%, 97%, 98% or 99% identical to a nucleic acid sequenceshown in SEQ ID NO:1 or SEQ ID NO:4, or to a nucleic acid sequence ofthe deposited cDNA which do, in fact, encode a polypeptide havingTACE-like or matrilysin-like protein activity. By “a polypeptide havingTACE-like activity” is intended polypeptides exhibiting TACE-likeactivity in a particular biological assay. For example, TACE-likeprotein activity can be measured using an assay for in vitro TNF-alphaprecursor cleavage, as described in Robache-Gallea, S. et al. J. Biol.Chem.270:23688-23692 (October 1995). By “a polypeptide havingmatrilysin-like activity” is intended polypeptides exhibitingmatrilysin-like activity in a particular biological assay. For example,matrilysin-like protein activity can be measured using the assaysdescribed in C. L. Wilson et al., Int. J. Biochem. Cell. Biol.28:123-136 (1996) or C. L. Wilson et al., Proc. Natl. Acad. Sci. USA94:1402-1407 (February 1997).

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%,98%, or 99% identical to a nucleic acid sequence of the deposited cDNAor a nucleic acid sequence shown in SEQ ID NO:1 or SEQ ID NO:4 willencode a polypeptide “having TACE-like or matrilysin-like proteinactivity.” In fact, since degenerate variants of these nucleotidesequences all encode the same polypeptide, this will be clear to theskilled artisan even without performing the above described comparisonassay. It will be further recognized in the art that, for such nucleicacid molecules that are not degenerate variants, a reasonable numberwill also encode a polypeptide having TACE-like or matrilysin-likeprotein activity. This is because the skilled artisan is fully aware ofamino acid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid).

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in J. U. Bowie et al., “Deciphering theMessage in Protein Sequences: Tolerance to Amino Acid Substitutions,”Science 247:1306-1310 (1990), wherein the authors indicate that proteinsare surprisingly tolerant of amino acid substitutions.

Vectors and Host Cells

The present invention also relates to vectors which include the isolatedDNA molecules of the present invention, host cells which are geneticallyengineered with the recombinant vectors, and the production of TACE-likeand matrilysin-like polypeptides or fragments thereof by recombinanttechniques.

The polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The DNA insert should be operatively linked to an appropriate promoter,such as the phage lambda PL promoter, the E. coli lac, trp and tacpromoters, the SV40 early and late promoters and promoters of retroviralLTRs, to name a few. Other suitable promoters will be known to theskilled artisan. The expression constructs will further contain sitesfor transcription initiation, termination and, in the transcribedregion, a ribosome binding site for translation. The coding portion ofthe mature transcripts expressed by the constructs will preferablyinclude a translation initiating at the beginning and a terminationcodon (UAA, UGA or UAG) appropriately positioned at the end of thepolypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase orneomycin resistance for eukaryotic cell culture and tetracycline orampicillin resistance genes for culturing in E. coli and other bacteria.Representative examples of appropriate hosts include, but are notlimited to, bacterial cells, such as E. coli, Streptomyces andSalmonella typhimurium cells; fungal cells, such as yeast cells; insectcells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells suchas CHO, COS and Bowes melanoma cells; and plant cells. Appropriateculture mediums and conditions for the above-described host cells areknown in the art.

Among vectors preferred for use in bacteria include pQE70, pQE60 andpQE-9, available from Qiagen; pBS vectors, Phagescript vectors,Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46A, available fromStratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia. Among preferred eukaryotic vectors are pWLNEO, pSV2CAT,pOG44, pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG andpSVL available from Pharmacia. Other suitable vectors will be readilyapparent to the skilled artisan.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986).

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals, but also additionalheterologous functional regions. For instance, a region of additionalamino acids, particularly charged amino acids, may be added to theN-terminus of the polypeptide to improve stability and persistence inthe host cell, during purification, or during subsequent handling andstorage. Also, peptide moieties may be added to the polypeptide tofacilitate purification. Such regions may be removed prior to finalpreparation of the polypeptide. The addition of peptide moieties topolypeptides to engender secretion or excretion, to improve stabilityand to facilitate purification, among others, are familiar and routinetechniques in the art. A preferred fusion protein comprises aheterologous region from immunoglobulin that is useful to solubilizeproteins. For example, EP-A-O 464 533 (Canadian counterpart 2045869)discloses fusion proteins comprising various portions of constant regionof immunoglobin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is thoroughlyadvantageous for use in therapy and diagnosis and thus results, forexample, in improved pharmacokinetic properties (EP-A 0232 262). On theother hand, for some uses it would be desirable to be able to delete theFc part after the fusion protein has been expressed, detected andpurified in the advantageous manner described. This is the case when Fcportion proves to be a hindrance to use in therapy and diagnosis, forexample when the fusion protein is to be used as an antigen forimmunizations. In drug discovery, for example, human proteins, such as,hIL5-receptor has been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. See,D. Bennett et al., Journal of Molecular Recognition 8:52-58 (1995) andK. Johanson et al., The Journal of Biological Chemistry 270:9459-9471(1995).

The TACE-like or matrilysin-like protein can be recovered and purifiedfrom recombinant cell cultures by well-known methods including ammoniumsulfate or ethanol precipitation, acid extraction, anion or cationexchange chromatography, phosphocellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. Most preferably, highperformance liquid chromatography (“HPLC”) is employed for purification.Polypeptides of the present invention include naturally purifiedproducts, products of chemical synthetic procedures, and productsproduced by recombinant techniques from a prokaryotic or eukaryotichost, including, for example, bacterial, yeast, higher plant, insect andmammalian cells. Depending upon the host employed in a recombinantproduction procedure, the polypeptides of the present invention may beglycosylated or may be non-glycosylated. In addition, polypeptides ofthe invention may also include an initial modified methionine residue,in some cases as a result of host-mediated processes.

TACE-Like and Matrilysin-Like Polypeptides and Fragments

The invention further provides an isolated TACE-like or matrilysin-likepolypeptide having the amino acid sequence encoded by the depositedcDNAs, or the amino acid sequence in SEQ ID NO:2 or SEQ ID NO:5, or apeptide or polypeptide comprising a portion of the above polypeptides.

It will be recognized in the art that some amino acid sequences of theTACE-like or matrilysin-like polypeptide can be varied withoutsignificant effect on the structure or function of the protein. If suchdifferences in sequence are contemplated, it should be remembered thatthere will be critical areas on the protein which determine activity.

Thus, the invention further includes variations of the TACE-like ormatrilysin-like polypeptide which show substantial TACE-like ormatrilysin-like polypeptide activity or which include regions ofTACE-like or matrilysin-like protein such as the protein portionsdiscussed below. Such mutants include deletions, insertions, inversions,repeats, and type substitutions. As indicated above, guidance concerningwhich amino acid changes are likely to be phenotypically silent can befound in J. U. Bowie et al., “Deciphering the Message in ProteinSequences: Tolerance to Amino Acid Substitutions,” Science 247:1306-1310(1990).

Thus, the fragment, derivative or analog of the polypeptide of SEQ IDNO:2 or SEQ ID NO:5, or that encoded by the deposited cDNA, may be (i)one in which one or more of the amino acid residues are substituted witha conserved or non-conserved amino acid residue (preferably a conservedamino acid residue) and such substituted amino acid residue may or maynot be one encoded by the genetic code, or (ii) one in which one or moreof the amino acid residues includes a substituent group, or (iii) one inwhich the mature polypeptide is fused with another compound, such as acompound to increase the half-life of the polypeptide (for example,polyethylene glycol), or (iv) one in which the additional amino acidsare fused to the mature polypeptide, such as an IgG Fc fusion regionpeptide or leader or secretory sequence or a sequence which is employedfor purification of the mature polypeptide or a proprotein sequence.Such fragments, derivatives and analogs are deemed to be within thescope of those skilled in the art from the teachings herein.

Of particular interest are substitutions of charged amino acids withanother charged amino acid and with neutral or negatively charged aminoacids. The latter results in proteins with reduced positive charge toimprove the characteristics of the TACE-like or matrilysin-like protein.The prevention of aggregation is highly desirable. Aggregation ofproteins not only results in a loss of activity but can also beproblematic when preparing pharmaceutical formulations, because they canbe immunogenic. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36:838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993)).

The replacement of amino acids can also change the selectivity ofbinding to cell surface receptors. Ostade et al., Nature 361:266-268(1993) describes certain mutations resulting in selective binding ofTNF-α to only one of the two known types of TNF receptors. Thus, themetalloproteinases of the present invention may include one or moreamino acid substitutions, deletions or additions, either from naturalmutations or human manipulation.

As indicated, changes are preferably of a minor nature, such asconservative amino acid substitutions that do not significantly affectthe folding or activity of the protein (see Table 1).

TABLE 1 Conservative Amino Acid Substitutions Aromatic PhenylalanineTryptophan Tyrosine Hydrophobic Leucine Isoleucine Valine PolarGlutamine Asparagine Basic Arginine Lysine Histidine Acidic AsparticAcid Glutamic Acid Small Alanine Serine Threonine Methionine Glycine

Amino acids in the TACE-like and matrilysin-like protein of the presentinvention that are essential for function can be identified by methodsknown in the art, such as site-directed mutagenesis or alanine-scanningmutagenesis (Cunningham and Wells, Science 244:1081-1085 (1989)). Thelatter procedure introduces single alanine mutations at every residue inthe molecule. The resulting mutant molecules are then tested forbiological activity such as receptor binding or in vitro, or in vitroproliferative activity. Sites that are critical for ligand-receptorbinding can also be determined by structural analysis such ascrystallization, nuclear magnetic resonance or photoaffinity labeling(Smith et al., J. Mol. Biol. 224:899-904 (1992) and de Vos et al.Science 255:306-312 (1992)). The polypeptides of the present inventionare preferably provided in an isolated form. By “isolated polypeptide”is intended a polypeptide removed from its native environment. Thus, apolypeptide produced and/or contained within a recombinant host cell isconsidered isolated for purposes of the present invention. Also intendedas an “isolated polypeptide” are polypeptides that have been purified,partially or substantially, from a recombinant host cell. For example, arecombinantly produced version of the TACE-like or matrilysin-likepolypeptide can be substantially purified by the one-step methoddescribed in Smith and Johnson, Gene 67:31-40 (1988).

The TACE-like polypeptides of the present invention include thepolypeptide encoded by the deposited cDNA including the leader; themature polypeptide encoded by the deposited the cDNA minus the leader(i.e., the mature protein); a polypeptide comprising amino acids about 1to about 182 in SEQ ID NO:2; a polypeptide comprising amino acids about2 to about 182 in SEQ ID NO:2; a polypeptide comprising amino acidsabout 16 to about 182 in SEQ ID NO:2; a polypeptide comprising thetransmembrane domain; as well as polypeptides which are at least 80%identical, more preferably at least 90% or 95% identical, still morepreferably at least 96%, 97%, 98% or 99% identical to those describedabove and also include portions of such polypeptides with at least 30amino acids and more preferably at least 50 amino acids.

The matrilysin-like polypeptides of the present invention include thepolypeptide encoded by the deposited cDNA including the leader; themature polypeptide encoded by the deposited the cDNA minus the leader(i.e., the mature protein); a polypeptide comprising amino acids about 1to about 261 in SEQ ID NO:5; a polypeptide comprising amino acids about2 to about 261 in SEQ ID NO:5; a polypeptide comprising amino acidsabout 23 to about 261 in SEQ ID NO:5; as well as polypeptides which areat least 80% identical, more preferably at least 90% or 95% identical,still more preferably at least 96%, 97%, 98% or 99% identical to thosedescribed above and also include portions of such polypeptides with atleast 30 amino acids and more preferably at least 50 amino acids.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a reference amino acid sequence of a TACE-like ormatrilysin-like polypeptide is intended that the amino acid sequence ofthe polypeptide is identical to the reference sequence except that thepolypeptide sequence may include up to five amino acid alterations pereach 100 amino acids of the reference amino acid of the TACE-like ormatrilysin-like polypeptide. In other words, to obtain a polypeptidehaving an amino acid sequence at least 95% identical to a referenceamino acid sequence, up to 5% of the amino acid residues in thereference sequence may be deleted or substituted with another aminoacid, or a number of amino acids up to 5% of the total amino acidresidues in the reference sequence may be inserted into the referencesequence. These alterations of the reference sequence may occur at theamino or carboxy terminal positions of the reference amino acid sequenceor anywhere between those terminal positions, interspersed eitherindividually among residues in the reference sequence or in one or morecontiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the aminoacid sequence shown in SEQ ID NO:2 or SEQ ID NO:5, or to the amino acidsequence encoded by the deposited cDNA clones can be determinedconventionally using known computer programs such as the Bestfit program(Wisconsin Sequence Analysis Package, Version 8 for Unix, GeneticsComputer Group, University Research Park, 575 Science Drive, Madison,Wis. 53711). When using Bestfit or any other sequence alignment programto determine whether a particular sequence is, for instance, 95%identical to a reference sequence according to the present invention,the parameters are set, of course, such that the percentage of identityis calculated over the full length of the reference amino acid sequenceand that gaps in homology of up to 5% of the total number of amino acidresidues in the reference sequence are allowed.

The polypeptides of the present invention are useful as molecular weightmarkers on SDS-PAGE gels or on molecular sieve gel filtration columnsusing methods well known to those of skill in the art.

In another aspect, the invention provides a peptide or polypeptidecomprising an epitope-bearing portion of a polypeptide of the invention.The epitope of this polypeptide portion is an immunogenic or antigenicepitope of a polypeptide described herein. An “immunogenic epitope” isdefined as a part of a protein that elicits an antibody response whenthe whole protein is the immunogen. On the other hand, a region of aprotein molecule to which an antibody can bind is defined as an“antigenic epitope.” The number of immunogenic epitopes of a proteingenerally is less than the number of antigenic epitopes. See, forinstance, Geysen et al., Proc. Natl. Acad. Sci. USA 81:3998-4002 (1983).

As to the selection of peptides or polypeptides bearing an antigenicepitope (i.e., that contain a region of a protein molecule to which anantibody can bind), it is well known in that art that relatively shortsynthetic peptides that mimic part of a protein sequence are routinelycapable of eliciting an antiserum that reacts with the partiallymimicked protein. See, for instance, Sutcliffe, J. G., Shinnick, T. M.,Green, N. and Learner, R. A., “Antibodies That React With PredeterminedSites on Proteins,” Science 219:660-666 (1983). Peptides capable ofeliciting protein-reactive sera are frequently represented in theprimary sequence of a protein, can be characterized by a set of simplechemical rules, and are confined neither to immunodominant regions ofintact proteins (i.e., immunogenic epitopes) nor to the amino orcarboxyl terminals.

Antigenic epitope-bearing peptides and polypeptides of the invention aretherefore useful to raise antibodies, including monoclonal antibodies,that bind specifically to a polypeptide of the invention. See, forinstance, Wilson et al., Cell 37:767-778 (1984) at 777.

Antigenic epitope-bearing peptides and polypeptides of the inventionpreferably contain a sequence of at least seven, more preferably atleast nine and most preferably between about at least about 15 to about30 amino acids contained within the amino acid sequence of a polypeptideof the invention.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate TACE-like-specific antibodies include: a polypeptidecomprising amino acid residues from about 13 to about 22 in SEQ ID NO:2;a polypeptide comprising amino acid residues from about 31 to about 46in SEQ ID NO:2; a polypeptide comprising amino acid residues from about55 to about 103 in SEQ ID NO:2; a polypeptide comprising amino acidresidues from about 123 to about 151 in SEQ ID NO:2; and a polypeptidecomprising amino acid residues from about 159 to about 173 in SEQ ID NO2. As indicated above, the inventors have determined that the abovepolypeptide fragments are antigenic regions of the TACE-like protein.

Non-limiting examples of antigenic polypeptides or peptides that can beused to generate matrilysin-like-specific antibodies include: apolypeptide comprising amino acid residues from about 24 to about 71 inSEQ ID NO:5; a polypeptide comprising amino acid residues from about 81to about 125 in SEQ ID NO:5; a polypeptide comprising amino acidresidues from about 141 to about 167 in SEQ ID NO:5; a polypeptidecomprising amino acid residues from about 173 to about 202 in SEQ IDNO:5; and a polypeptide comprising amino acid residues from about 212 toabout 260 in SEQ ID NO:5. As indicated above, the inventors havedetermined that the above polypeptide fragments are antigenic regions ofthe matrilysin-like protein.

The epitope-bearing peptides and polypeptides of the invention may beproduced by any conventional means. Houghten, R. A., “General Method forthe Rapid Solid-Phase Synthesis of Large Numbers of Peptides:Specificity of Antigen-Antibody Interaction at the Level of IndividualAmino Acids, Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985). This“Simultaneous Multiple Peptide Synthesis (SMPS)” process is furtherdescribed in U.S. Pat. No. 4,631,211 to Houghten et al. (1986).

As one of skill in the art will appreciate, TACE-like or matrilysin-likepolypeptides of the present invention and the epitope-bearing fragmentsthereof described above can be combined with parts of the constantdomain of immunoglobulins (IgG), resulting in chimeric polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. This has been shown, e.g., for chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature331:84-86 (1988)). Fusion proteins that have a disulfide-linked dimericstructure due to the IgG part can also be more efficient in binding andneutralizing other molecules than the monomeric TACE-like ormatrilysin-like protein or protein fragment alone (Fountoulakis et al.,J. Biochem 270:3958-3964 (1995)).

Matrilysin-Like Protein in Cancer Diagnosis and Prognosis

The expression of matrix metalloproteinases (MMPs) in neoplastic lesionshas been well-documented. The mRNA of one MMP, matrilysin (“MMP-7”), hasbeen detected in human adenomas, as well as carcinomas andadenocarcinomas of the breast and colon. Heppner, K. J. et al., Am. J.Pathol. 149:273-282 (1996); Wolf, C. et al., Proc. Natl. Acad. Sci. USA90:1843-1847 (1993). MMP-7 has also been reported to be expressed inhuman gastric carcinomas. Honda, M. et al., Gut 39:444-448 (1996).Recently, Wilson, C. L. et al., Proc. Natl. Acad. Sci. USA 94:1402-1407(February 1997), has reported that matrilysin is expressed in a highpercentage of early-stage human colorectal tumors. Accordingly, it isbelieved that certain tissues in mammals with cancer expresssignificantly enhanced levels of the matrilysin-like protein and mRNAencoding the matrilysin-like protein when compared to a corresponding“standard” mammal, i.e., a mammal of the same species not having thecancer. Further, it is believed that enhanced levels of thematrilysin-like protein can be detected in certain body fluids (e.g.,sera, plasma, urine, and spinal fluid) from mammals with cancer whencompared to sera from mammals of the same species not having the cancer.Thus, the invention provides a diagnostic method useful during tumordiagnosis, which involves assaying the expression level of the geneencoding the matrilysin-like protein in mammalian cells or body fluidand comparing the gene expression level with a standard matrilysin-likegene expression level, whereby an increase in the gene expression levelover the standard is indicative of certain tumors.

Where a tumor diagnosis has already been made according to conventionalmethods, the present invention is useful as a prognostic indicator,whereby patients exhibiting enhanced matrilysin-like gene expressionwill experience a worse clinical outcome relative to patients expressingthe gene at a lower level.

By “assaying the expression level of the gene encoding thematrilysin-like protein” is intended qualitatively or quantitativelymeasuring or estimating the level of the matrilysin-like protein or thelevel of the mRNA encoding the matrilysin-like protein in a firstbiological sample either directly (e.g., by determining or estimatingabsolute protein level or mRNA level) or relatively (e.g., by comparingto the matrilysin-like protein level or mRNA level in a secondbiological sample).

Preferably, the matrilysin-like protein level or mRNA level in the firstbiological sample is measured or estimated and compared to a standardmatrilysin-like protein level or mRNA level, the standard being takenfrom a second biological sample obtained from an individual not havingthe cancer. As will be appreciated in the art, once a standardmatrilysin-like protein level or mRNA level is known, it can be usedrepeatedly as a standard for comparison.

By “biological sample” is intended any biological sample obtained froman individual, cell line, tissue culture, or other source which containsmatrilysin-like protein or mRNA. Biological samples include mammalianbody fluids (such as sera, plasma, urine, synovial fluid and spinalfluid) which contain secreted mature matrilysin-like protein, andovarian, prostate, heart, placenta, pancreas liver, spleen, lung, breastand umbilical tissue.

The present invention is useful for detecting cancer in mammals. Inparticular the invention is useful during diagnosis of the followingtypes of cancers in mammals: intestinal (colon), stomach, breast,ovarian, prostate, bone, liver, lung, pancreatic, and spleenic.Preferred mammals include monkeys, apes, cats, dogs, cows, pigs, horses,rabbits and humans. Particularly preferred are humans.

Total cellular RNA can be isolated from a biological sample using thesingle-step guanidinium-thiocyanate-phenol-chloroform method describedin Chomczynski and Sacchi, Anal. Biochem. 162:156-159 (1987). Levels ofmRNA encoding the matrilysin-like protein are then assayed using anyappropriate method. These include Northern blot analysis (Harada et al.,Cell 63:303-312 (1990)), S1 nuclease mapping (Fujita et al., Cell49:357-367 (1987)), the polymerase chain reaction (PCR), reversetranscription in combination with the polymerase chain reaction (RT-PCR)(Makino et al., Technique 2:295-301 (1990)), and reverse transcriptionin combination with the ligase chain reaction (RT-LCR).

Northern blot analysis can be performed as described in Harada et al.,Cell 63:303-312 (1990). Briefly, total RNA is prepared from a biologicalsample as described above. For the Northern blot, the RNA is denaturedin an appropriate buffer (such as glyoxal/dimethyl sulfoxide/sodiumphosphate buffer), subjected to agarose gel electrophoresis, andtransferred onto a nitrocellulose filter. After the RNAs have beenlinked to the filter by a UV linker, the filter is prehybridized in asolution containing formamide, SSC, Denhardt's solution, denaturedsalmon sperm, SDS, and sodium phosphate buffer. Matrilysin-like proteincDNA labeled according to any appropriate method (such as the³²P-multiprimed DNA labeling system (Amersham)) is used as probe. Afterhybridization overnight, the filter is washed and exposed to x-ray film.cDNA for use as probe according to the present invention is described inthe sections above and will preferably at least 15 bp in length.

S1 mapping can be performed as described in Fujita et al., Cell49:357-367 (1987). To prepare probe DNA for use in S1 mapping, the sensestrand of above-described cDNA is used as a template to synthesizelabeled antisense DNA. The antisense DNA can then be digested using anappropriate restriction endonuclease to generate further DNA probes of adesired length. Such antisense probes are useful for visualizingprotected bands corresponding to the target mRNA (i.e., mRNA encodingthe matrilysin-like protein). Northern blot analysis can be performed asdescribed above.

Preferably, levels of mRNA encoding the matrilysin-like protein areassayed using the RT-PCR method described in Makino et al., Technique2:295-301 (1990). By this method, the radioactivities of the “amplicons”in the polyacrylamide gel bands are linearly related to the initialconcentration of the target mRNA. Briefly, this method involves addingtotal RNA isolated from a biological sample in a reaction mixturecontaining a RT primer and appropriate buffer. After incubating forprimer annealing, the mixture can be supplemented with a RT buffer,dNTPs, DTT, RNase inhibitor and reverse transcriptase. After incubationto achieve reverse transcription of the RNA, the RT products are thensubject to PCR using labeled primers. Alternatively, rather thanlabeling the primers, a labeled dNTP can be included in the PCR reactionmixture. PCR amplification can be performed in a DNA thermal cycleraccording to conventional techniques. After a suitable number of roundsto achieve amplification, the PCR reaction mixture is electrophoresed ona polyacrylamide gel. After drying the gel, the radioactivity of theappropriate bands (corresponding to the mRNA encoding thematrilysin-like protein) is quantified using an imaging analyzer. RT andPCR reaction ingredients and conditions, reagent and gel concentrations,and labeling methods are well known in the art. Variations on the RT-PCRmethod will be apparent to the skilled artisan.

Any set of oligonucleotide primers which will amplify reversetranscribed target mRNA can be used and can be designed as described inthe sections above.

Assaying matrilysin-like protein levels in a biological sample can occurusing any art-known method. Preferred for assaying matrilysin-likeprotein levels in a biological sample are antibody-based techniques. Forexample, matrilysin-like protein expression in tissues can be studiedwith classical immunohistological methods. In these, the specificrecognition is provided by the primary antibody (polyclonal ormonoclonal) but the secondary detection system can utilize fluorescent,enzyme, or other conjugated secondary antibodies. As a result, animmunohistological staining of tissue section for pathologicalexamination is obtained. Tissues can also be extracted, e.g., with ureaand neutral detergent, for the liberation of matrilysin-like protein forWestern-blot or dot/slot assay (Jalkanen, M., et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, M., et al., J. Cell. Biol. 105:3087-3096(1987)). In this technique, which is based on the use of cationic solidphases, quantitation of matrilysin-like protein can be accomplishedusing isolated matrilysin-like protein as a standard. This technique canalso be applied to body fluids. With these samples, a molarconcentration of matrilysin-like protein will aid to set standard valuesof matrilysin-like protein content for different body fluids, likeserum, plasma, urine, spinal fluid, etc. The normal appearance ofmatrilysin-like protein amounts can then be set using values fromhealthy individuals, which can be compared to those obtained from a testsubject.

Other antibody-based methods useful for detecting matrilysin-likeprotein gene expression include immunoassays, such as the enzyme linkedimmunosorbent assay (ELISA) and the radioimmunoassay (RIA). For example,a matrilysin-like protein-specific monoclonal antibodies can be usedboth as an immunoabsorbent and as an enzyme-labeled probe to detect andquantify the matrilysin-like protein. The amount of matrilysin-likeprotein present in the sample can be calculated by reference to theamount present in a standard preparation using a linear regressioncomputer algorithm. Such an ELISA for detecting a tumor antigen isdescribed in Iacobelli et al., Breast Cancer Research and Treatment11:19-30 (1988). In another ELISA assay, two distinct specificmonoclonal antibodies can be used to detect matrilysin-like protein in abody fluid. In this assay, one of the antibodies is used as theimmunoabsorbent and the other as the enzyme-labeled probe.

The above techniques may be conducted essentially as a “one-step” or“two-step” assay. The “one-step” assay involves contactingmatrilysin-like protein with immobilized antibody and, without washing,contacting the mixture with the labeled antibody. The “two-step” assayinvolves washing before contacting the mixture with the labeledantibody. Other conventional methods may also be employed as suitable.It is usually desirable to immobilize one component of the assay systemon a support, thereby allowing other components of the system to bebrought into contact with the component and readily removed from thesample.

Suitable enzyme labels include, for example, those from the oxidasegroup, which catalyze the production of hydrogen peroxide by reactingwith substrate. Glucose oxidase is particularly preferred as it has goodstability and its substrate (glucose) is readily available. Activity ofan oxidase label may be assayed by measuring the concentration ofhydrogen peroxide formed by the enzyme-labeled antibody/substratereaction. Besides enzymes, other suitable labels include radioisotopes,such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulphur (³⁵S), tritium (³H),indium (¹¹²In), and technetium (^(99m)Tc), and fluorescent labels, suchas fluorescein and rhodamine, and biotin.

In addition to assaying matrilysin-like protein levels in a biologicalsample obtained from an individual, matrilysin-like protein can also bedetected in vivo by imaging. Antibody labels or markers for in vivoimaging of matrilysin-like protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A matrilysin-like protein-specific antibody or antibody fragment whichhas been labeled with an appropriate detectable imaging moiety, such asa radioisotope (for example, ¹³¹I, ¹¹²In, ^(99m)Tc), a radio-opaquesubstance, or a material detectable by nuclear magnetic resonance, isintroduced (for example, parenterally, subcutaneously orintraperitoneally) into the mammal to be examined for cancer. It will beunderstood in the art that the size of the subject and the imagingsystem used will determine the quantity of imaging moiety needed toproduce diagnostic images. In the case of a radioisotope moiety, for ahuman subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled antibodyor antibody fragment will then preferentially accumulate at the locationof cells which contain matrilysin-like protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabelled Antibodies and Their Fragments” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982)).

Matrilysin-like-protein specific antibodies for use in the presentinvention can be raised against the intact matrilysin-like protein or anantigenic polypeptide fragment thereof, which may presented togetherwith a carrier protein, such as an albumin, to an animal system (such asrabbit or mouse) or, if it is long enough (at least about 25 aminoacids), without a carrier.

As used herein, the term “antibody” (Ab) or “monoclonal antibody” (Mab)is meant to include intact molecules as well as antibody fragments (suchas, for example, Fab and F(ab′)₂ fragments) that are capable ofspecifically binding to matrilysin-like protein. Fab and F(ab′)₂fragments lack the Fc fragment of intact antibody, clear more rapidlyfrom the circulation, and may have less non-specific tissue binding ofan intact antibody (Wahl et al., J. Nucl. Med. 24:316-325 (1983)). Thus,these fragments are preferred.

The antibodies of the present invention may be prepared by any of avariety of methods. For example, cells expressing the matrilysin-likeprotein or an antigenic fragment thereof can be administered to ananimal in order to induce the production of sera containing polyclonalantibodies. In a preferred method, a preparation of matrilysin-likeprotein is prepared and purified to render it substantially free ofnatural contaminants. Such a preparation is then introduced into ananimal in order to produce polyclonal antisera of greater specificactivity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or matrilysin-like protein binding fragmentsthereof). Such monoclonal antibodies can be prepared using hybridomatechnology (Kohier et al., Nature 256:495 (1975); Kohler et al., Eur. J.Immunol. 6:511(1976); Kohler et al., Eur. J. Immunol. 6:292 (1976);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas,Elsevier, N.Y., (1981) pp. 563-681). In general, such procedures involveimmunizing an animal (preferably a mouse) with a matrilysin-like proteinantigen or, more preferably, with a matrilysin-like protein-expressingcell. Suitable cells can be recognized by their capacity to bindanti-matrilysin-like protein antibody. Such cells may be cultured in anysuitable tissue culture medium; however, it is preferable to culturecells in Earle's modified Eagle's medium supplemented with 10% fetalbovine serum (inactivated at about 56° C.), and supplemented with about10 g/l of nonessential amino acids, about 1,000 U/ml of penicillin, andabout 100 μg/ml of streptomycin. The splenocytes of such mice areextracted and fused with a suitable mycloma cell line. Any suitablemyeloma cell line may be employed in accordance with the presentinvention; however, it is preferable to employ the parent myeloma cellline (SP₂O), available from the American Type Culture Collection,Manassas, Md. After fusion, the resulting hybridoma cells areselectively maintained in HAT medium, and then cloned by limitingdilution as described by Wands et al. (Gastroenterology 80:225-232(1981)). The hybridoma cells obtained through such a selection are thenassayed to identify clones which secrete antibodies capable of bindingthe matrilysin-like protein antigen.

Alternatively, additional antibodies capable of binding to thematrilysin-like protein antigen may be produced in a two-step procedurethrough the use of anti-idiotypic antibodies. Such a method makes use ofthe fact that antibodies are themselves antigens, and that, therefore,it is possible to obtain an antibody which binds to a second antibody.In accordance with this method, matrilysin-like-protein specificantibodies are used to immunize an animal, preferably a mouse. Thesplenocytes of such an animal are then used to produce hybridoma cells,and the hybridoma cells are screened to identify clones which produce anantibody whose ability to bind to the matrilysin-like protein-specificantibody can be blocked by the matrilysin-like protein antigen. Suchantibodies comprise anti-idiotypic antibodies to the matrilysin-likeprotein-specific antibody and can be used to immunize an animal toinduce formation of further matrilysin-like protein-specific antibodies.

It will be appreciated that Fab and F(ab′)₂ and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). Alternatively, matrilysin-likeprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

Where in vivo imaging is used to detect enhanced levels ofmatrilysin-like protein for tumor diagnosis in humans, it may bepreferable to use “humanized” chimeric monoclonal antibodies. Suchantibodies can be produced using genetic constructs derived fromhybridoma cells producing the monoclonal antibodies described above.Methods for producing chimeric antibodies are known in the art. See, forreview, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques4:214 (1986); Cabilly et al., U.S. Pat. No. 4,816,567; Taniguchi et al.,EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;Robinson et al., WO 8702671; Boulianne et al., Nature 312:643 (1984);Neuberger et al., Nature 314:268 (1985).

Further suitable labels for the matrilysin-like protein-specificantibodies of the present invention are provided below. Examples ofsuitable enzyme labels include malate dehydrogenase, staphylococcalnuclease, delta-5-steroid isomerase, yeast-alcohol dehydrogenase,alpha-glycerol phosphate dehydrogenase, triose phosphate isomerase,peroxidase, alkaline phosphatase, asparaginase, glucose oxidase,beta-galactosidase, ribonuclease, urease, catalase, glucose-6-phosphatedehydrogenase, glucoamylase, and acetylcholine esterase.

Examples of suitable radioisotopic labels include ³H, ¹¹¹In, ¹²⁵I, ¹³¹I,³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci,²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd, etc. ¹¹¹In is a preferred isotope where invivo imaging is used since its avoids the problem of dehalogenation ofthe ¹²⁵I or ¹³¹I-labeled monoclonal antibody by the liver. In addition,this radionucleotide has a more favorable gamma emission energy forimaging (Perkins et al., Eur. J. Nucl. Med. 10:296-301 (1985);Carasquillo et al., J. Nucl. Med. 28:281-287 (1987)). For example, ¹¹¹Incoupled to monoclonal antibodies with 1-(P-isothiocyanatobenzyl)-DPTAhas shown little uptake in non-tumorous tissues, particularly the liver,and therefore enhances specificity of tumor localization (Esteban etal., J. Nucl. Med. 28:861-870 (1987)).

Examples of suitable non-radioactive isotopic labels include ¹⁵⁷Gd,⁵⁵Mn, ¹⁶²Dy, ⁵²Tr, and ⁵⁶Fe.

Examples of suitable fluorescent labels include an ¹⁵²Eu label, afluorescein label, an isothiocyanate label, a rhodamine label, aphycoerythrin label, a phycocyanin label, an allophycocyanin label, ano-phthaldehyde label, and a fluorescamine label.

Examples of suitable toxin labels include diphtheria toxin, ricin, andcholera toxin.

Examples of chemiluminescent labels include a luminal label, anisoluminal label, an aromatic acridinium ester label, an imidazolelabel, an acridinium salt label, an oxalate ester label, a luciferinlabel, a luciferase label, and an aequorin label.

Examples of nuclear magnetic resonance contrasting agents include heavymetal nuclei such as Gd, Mn, and iron.

Typical techniques for binding the above-described labels to antibodiesare provided by Kennedy et al., Clin. Chim. Acta 70:1-31 (1976), andSchurs et al., Clin. Chim. Acta 81:1-40 (1977). Coupling techniquesmentioned in the latter are the glutaraldehyde method, the periodatemethod, the dimaleimide method, them-maleimidobenzyl-N-hydroxy-succinimide ester method, all of whichmethods are incorporated by reference herein.

Screening for Antagonists and Agonists

The present invention also provides a method of screening compounds toidentify those which enhance or block the action of the TACE-like ormatrilysin-like protein on cells, such as its interaction with moleculesthat bind TACE-like or matrilysin-like proteins. An agonist is acompound which increases the natural biological functions of TACE-likeor matrilysin-like protein, or which functions in a manner similar toTACE-like or matrilysin-like protein, while antagonists decrease oreliminate such functions. For example, a cellular compartment such as amembrane or a preparation thereof may be prepared from a cell thatexpresses a molecule that binds TACE-like or matrilysin-like proteins.The preparation is then incubated with labeled TACE-like ormatrilysin-like protein in the absence of or presence of a candidatecompound which may be a TACE-like or matrilysin-like antagonist oragonist. The ability of the candidate compound to bind the bindingmolecule is reflected in decreased binding of the labeled ligand.Compounds which bind gratuitously (i.e., without inducing the effects ofTACE-like or matrilysin-like proteins on binding the TACE-like ormatrilysin-like binding molecule) are most likely good antagonists.Compounds that bind well and elicit effect that are the same as orclosely related to TACE-like or matrilysin-like proteins are agonists.

In another aspect, a screening assay for agonists and antagonists isprovided which involves determining the effect a candidate compound hason TACE-like binding to the TNF-alpha precursor. In particular, themethod involves contacting the TNF-alpha precursor with a TACE-likepolypeptide and a candidate compound and determining whether TACE-likepolypeptide binding to the TNF-alpha precursor is increased or decreaseddue to the presence of the candidate compound.

Potential antagonists include small organic molecules, peptides,polypeptides, and antibodies that bind to a polypeptide of the inventionand thereby inhibitits activity. Potential antagonists also includeantisense molecules For a review, see, Okano, J., J. Neurochem. 56:560(1991).

Agonists of the TACE-like protein of the invention may be used toenhance the action of TACE-like proteins, for example, in the treatmentof cancer, or any disease characterized by an underproduction ofTNF-alpha. Antagonists of the TACE-like protein of the invention may beused to inhibit the action of TACE-like proteins, for example, in thetreatment of disorders characterized by an overproduction of TNF-alpha,such as inflammation, immune system disorders, infectious disease, orneurological disease.

Antagonists of the matrilysin-like protein of the invention may be usedto inhibit the action of the matrilysin-like protein of the invention,for example, in the treatment or prophylaxis of disorders characterizedby degradation of the extracellular matrix, such as, for example,cancer, arthritis, cardiovascular disorders, cachexia, and multiplesclerosis.

The agonists and antagonists described herein may be employed in acomposition with a pharmaceutically acceptable carrier, as describedbelow.

Therapeutics

As discussed above, the TACE-like protein of the invention or agoniststhereof may be used in the treatment of cancer, or any diseasecharacterized by an underproduction of TNF-alpha. Antagonists of theTACE-like protein of the invention may be used to inhibit the action ofTACE-like proteins, for example, in the treatment of disorderscharacterized by an overproduction of TNF-alpha, such as inflammation,immune system disorders, infectious disease, or neurological disease.

Matrix metalloproteinase (MMP) inhibitors, such as an antagonist of thematrilysin-like protein of the invention, may be used to inhibit theaction of matrilysin-like proteins, for example, in the treatment ofdisorders characterized by degradation of the extracellular matrix, suchas, e.g, cancer, arthritis, cardiovascular disorders, cachexia, immunesystem disorders, digestive disorders and multiple sclerosis.

Modes of Administration

It will be appreciated that conditions caused by a decrease in thestandard or normal level of TACE-like activity in an individual, can betreated by administration of the TACE-like protein. Thus, the inventionfurther provides a method of treating an individual in need of anincreased level of TACE-like activity comprising administering to suchan individual a pharmaceutical composition comprising an effectiveamount of an isolated TACE-like polypeptide of the invention,particularly a mature form of the TACE-like protein, effective toincrease the TACE-like activity level in such an individual.

As a general proposition, the total pharmaceutically effective amount ofTACE-like polypeptide administered parenterally per dose will be in therange of about 1 μg/kg/day to 10 mg/kg/day of patient body weight,although, as noted above, this will be subject to therapeuticdiscretion. More preferably, this dose is at least 0.01 mg/kg/day, andmost preferably for humans between about 0.01 and 1 mg/kg/day for thehormone. If given continuously, the TACE-like polypeptide is typicallyadministered at a dose rate of about 1 μg/kg/hour to about 50μg/kg/hour, either by 1-4 injections per day or by continuoussubcutaneous infusions, for example, using a mini-pump. An intravenousbag solution may also be employed.

Pharmaceutical compositions containing the TACE-like protein of theinvention may be administered orally, rectally, parenterally,intracistemally, intravaginally, intraperitoneally, topically (as bypowders, ointments, drops or transdermal patch), bucally, or as an oralor nasal spray. By “pharmaceutically acceptable carrier” is meant anon-toxic solid, semisolid or liquid filler, diluent, encapsulatingmaterial or formulation auxiliary of any type. The term “parenteral” asused herein refers to modes of administration which include intravenous,intramuscular, intraperitoneal, intrasternal, subcutaneous andintraarticular injection and infusion.

Chromosome Assays

The nucleic acid molecules of the present invention are also valuablefor chromosome identification. The sequence is specifically targeted toand can hybridize with a particular location on an individual humanchromosome. The mapping of DNAs to chromosomes according to the presentinvention is an important first step in correlating those sequences withgenes associated with disease.

In certain preferred embodiments in this regard, the cDNA hereindisclosed is used to clone genomic DNA of a TACE-like or matrilysin-likeprotein gene. This can be accomplished using a variety of well knowntechniques and libraries, which generally are available commercially.The genomic DNA then is used for in situ chromosome mapping using wellknown techniques for this purpose.

In addition, in some cases, sequences can be mapped to chromosomes bypreparing PCR primers (preferably 15-25 bp) from the cDNA. Computeranalysis of the 3 untranslated region of the gene is used to rapidlyselect primers that do not span more than one exon in the genomic DNA,thus complicating the amplification process. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes.

Fluorescence in situ hybridization (“FISH”) of a cDNA clone to ametaphase chromosomal spread can be used to provide a precisechromosomal location in one step. This technique can be used with probesfrom the cDNA as short as 50 or 60 bp. For a review of this technique,see Verma et al., Human Chromosomes: A Manual Of Basic Techniques,Pergamon Press, New York (1988).

Once a sequence has been mapped to a precise chromosomal location, thephysical position of the sequence on the chromosome can be correlatedwith genetic map data. Such data are found, for example, in V. McKusick,Mendelian Inheritance In Man, available on-line through Johns HopkinsUniversity, Welch Medical Library. The relationship between genes anddiseases that have been mapped to the same chromosomal region are thenidentified through linkage analysis (coinheritance of physicallyadjacent genes).

Next, it is necessary to determine the differences in the cDNA orgenomic sequence between affected and unaffected individuals. If amutation is observed in some or all of the affected individuals but notin any normal individuals, then the mutation is likely to be thecausative agent of the disease.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1a Expression and Purification of TACE-Like Protein inE. coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). pQE60 encodes ampicillin antibiotic resistance (“Amp^(r)”) andcontains a bacterial origin of replication (“ori”), an IPTG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that a DNA fragment encoding apolypeptide may be inserted in such as way as to produce thatpolypeptide with the six His residues (i.e., a “6 X His tag”) covalentlylinked to the carboxyl terminus of that polypeptide. However, in thisexample, the polypeptide coding sequence is inserted such thattranslation of the six His codons is prevented and, therefore, thepolypeptide is produced with no 6 X His tag.

The DNA sequence encoding the desired portion of the TACE-like proteinlacking the hydrophobic leader sequence is amplified from the depositedcDNA clone using PCR oligonucleotide primers which anneal to the aminoterminal sequences of the desired portion of the TACE-like protein andto sequences in the deposited construct 3′ to the cDNA coding sequence.Additional nucleotides containing restriction sites to facilitatecloning in the pQE60 vector are added to the 5′ and 3′ sequences,respectively.

For cloning the mature protein, the 5′ primer has the sequence:

5′ CGCCCATGGCCCACGAAGGTTCTGAA 3′ (SEQ ID NO:10) containing theunderlined NcoI restriction site followed by 17 nucleotidescomplementary to the amino terminal coding sequence of the matureTACE-like sequence in FIG. 1. One of ordinary skill in the art wouldappreciate, of course, that the point in the protein coding sequencewhere the 5′ primer begins may be varied to amplify a desired portion ofthe complete protein shorter or longer than the mature form. The 3′primer has the sequence:

5′ CGCAAGCTTTCATTTGTAGGGCTGGTCTTTC 3′ (SEQ ID NO:11) containing theunderlined Hind III restriction site followed by 22 nucleotidescomplementary to the 3′ end of the non-coding sequence in the TACE-likeDNA sequence in FIG. 1.

The amplified TACE-like DNA fragments and the vector pQE60 are digestedwith NcoO and Hind III and the digested DNAs are then ligated together.Insertion of the TACE-like DNA into the restricted pQE60 vector placesthe TACE-like protein coding region including its associated stop codondownstream from the IPTG-inducible promoter and in-frame with aninitiating AUG. The associated stop codon prevents translation of thesix histidine codons downstream of the insertion point.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance(“Kan^(r)”), is used in carrying out the illustrative example describedherein. This strain, which is only one of many that are suitable forexpressing TACE-like protein, is available commercially from QIAGEN,Inc., supra. Transformants are identified by their ability to grow on LBplates in the presence of ampicillin and kanamycin. Plasmid DNA isisolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3-4 hours at 4 C. in 6M guanidine-HCl,pH8. The cell debris is removed by centrifugation, and the supernatantcontaining the TACE-like protein is dialyzed against 50 mM Na-acetatebuffer pH6, supplemented with 200 mM NaCl. Alternatively, the proteincan be successfully refolded by dialyzing it against 500 mM NaCl, 20%glycerol, 25 mM Tris/HCl pH7.4, containing protease inhibitors. Afterrenaturation the protein can be purified by ion exchange, hydrophobicinteraction and size exclusion chromatography. Alternatively, anaffinity chromatography step such as an antibody column can be used toobtain pure TACE-like protein. The purified protein is stored at 4 C orfrozen at −80 C.

Example 1b Expression and Purification of Matrilysin-Like Protein in E.coli

The bacterial expression vector pQE60 is used for bacterial expressionin this example. (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif.,91311). pQE60 encodes ampicillin antibiotic resistance (“Amp^(r)”) andcontains a bacterial origin of replication (“ori”), an IPTG induciblepromoter, a ribosome binding site (“RBS”), six codons encoding histidineresidues that allow affinity purification usingnickel-nitrilo-tri-acetic acid (“Ni-NTA”) affinity resin sold by QIAGEN,Inc., supra, and suitable single restriction enzyme cleavage sites.These elements are arranged such that a DNA fragment encoding apolypeptide may be inserted in such as way as to produce thatpolypeptide with the six His residues (i.e., a “6 X His tag”) covalentlylinked to the carboxyl terminus of that polypeptide. However, in thisexample, the polypeptide coding sequence is inserted such thattranslation of the six His codons is prevented and, therefore, thepolypeptide is produced with no 6 X His tag.

The DNA sequence encoding the desired portion of the matrilysin-likeprotein lacking the hydrophobic leader sequence is amplified from thedeposited cDNA clone using PCR oligonucleotide primers which anneal tothe amino terminal sequences of the desired portion of thematrilysin-like protein and to sequences in the deposited construct 3′to the cDNA coding sequence. Additional nucleotides containingrestriction sites to facilitate cloning in the pQE60 vector are added tothe 5′ and 3′ sequences, respectively.

For cloning the mature protein, the 5′ primer has the sequence:

5′ CGCCCATGGCTGCAGACCATAAAGGATG 3′ (SEQ ID NO:12) containing theunderlined NcoI restriction site followed by 19 nucleotidescomplementary to the amino terminal coding sequence of the maturematrilysin-like sequence in FIGS. 4A-B. One of ordinary skill in the artwould appreciate, of course, that the point in the protein codingsequence where the 5′ primer begins may be varied to amplify a desiredportion of the complete protein shorter or longer than the mature form.

The 3′ primer has the sequence:

5′ CGCAAGCTTCTCTAGTGCTTTCAGTTC 3′ (SEQ ID NO:13) containing theunderlined Hind III restriction site followed by 18 nucleotidescomplementary to the 3′ end of the non-coding sequence in thematrilysin-like DNA sequence in FIGS. 4A-B.

The amplified matrilysin-like DNA fragments and the vector pQE60 aredigested with NcoI and HindIII and the digested DNAs are then ligatedtogether. Insertion of the matrilysin-like DNA into the restricted pQE60vector places the matrilysin-like protein coding region including itsassociated stop codon downstream from the IPTG-inducible promoter andin-frame with an initiating AUG. The associated stop codon preventstranslation of the six histidine codons downstream of the insertionpoint.

The ligation mixture is transformed into competent E. coli cells usingstandard procedures such as those described in Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2nd Ed.; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y. (1989). E. coli strainM15/rep4, containing multiple copies of the plasmid pREP4, whichexpresses the lac repressor and confers kanamycin resistance(“Kan^(r)”), is used in carrying out the illustrative example describedherein. This strain, which is only one of many that are suitable forexpressing matrilysin-like protein, is available commercially fromQIAGEN, Inc., supra. Transformants are identified by their ability togrow on LB plates in the presence of ampicillin and kanamycin. PlasmidDNA is isolated from resistant colonies and the identity of the clonedDNA confirmed by restriction analysis, PCR and DNA sequencing.

Clones containing the desired constructs are grown overnight (“O/N”) inliquid culture in LB media supplemented with both ampicillin (100 μg/ml)and kanamycin (25 μg/ml). The O/N culture is used to inoculate a largeculture, at a dilution of approximately 1:25 to 1:250. The cells aregrown to an optical density at 600 nm (“OD600”) of between 0.4 and 0.6.Isopropyl-b-D-thiogalactopyranoside (“IPTG”) is then added to a finalconcentration of 1 mM to induce transcription from the lac repressorsensitive promoter, by inactivating the lacI repressor. Cellssubsequently are incubated further for 3 to 4 hours. Cells then areharvested by centrifugation.

The cells are then stirred for 3-4 hours at 4 C. in 6M guanidine-HCl,pH8. The cell debris is removed by centrifugation, and the supernatantcontaining the matrilysin-like protein is dialyzed against 50 mMNa-acetate buffer pH6, supplemented with 200 mM NaCl. Alternatively, theprotein can be successfully refolded by dialyzing it against 500 mMNaCl, 20% glycerol, 25 mM Tris/HCl pH7.4, containing proteaseinhibitors. After renaturation the protein can be purified by ionexchange, hydrophobic interaction and size exclusion chromatography.Alternatively, an affinity chromatography step such as an antibodycolumn can be used to obtain pure matrilysin-like protein. The purifiedprotein is stored at 4 C. or frozen at −80 C.

Example 2a Cloning and Expression of TACE-Like Protein in a BaculovirusExpression System

In this illustrative example, the plasmid shuttle vector pA2 is used toinsert the cloned DNA encoding the complete protein, including itsnaturally associated secretary signal (leader) sequence, into abaculovirus to express the mature TACE-like protein, using standardmethods as described in Summers et al., A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI and Asp7l8. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpress the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

The cDNA sequence encoding the full length TACE-like protein in thedeposited clone, including the AUG initiation codon and the naturallyassociated leader sequence shown in FIG. 1 (SEQ ID NO:1), is amplifiedusing PCR oligonucleotide primers corresponding to the 5′ and 3′sequences of the gene.

The 5′ primer has the sequence:

5′CGCGGATCCGCCATCATGTTCCTTCTCCTCGC 3′ (SEQ ID NO:14) containing theunderlined BamHI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by 17 bases (i.e., 22-38)of the sequence of the complete TACE-like protein shown in FIG. 1,beginning with the AUG initiation codon.

The 3′ primer (full length) has the sequence:

5′ CGCGGTACCACTACACAGGCAATGT 3′ (SEQ ID NO: 15) containing theunderlined, Asp718 restriction site followed by 16 nucleotides (i.e.,580-595) complementary to the 3′ noncoding sequence in FIG. 1.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and Asp718 and againis purified on a 1% agarose gel. This fragment is designated herein“F1”.

The plasmid is digested with the restriction enzymes BamHI and Asp718and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

Fragment F1 and the dephosphorylated plasmid V1 are ligated togetherwith T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts suchas XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria are identified that contain the plasmid with the humanTACE-like gene using the PCR method, in which one of the primers that isused to amplify the gene and the second primer is from well within thevector so that only those bacterial colonies containing the TACE-likegene fragment will show amplification of the DNA. The sequence of thecloned fragment is confirmed by DNA sequencing. This plasmid isdesignated herein pBacTACE-like.

Five μg of the plasmid pBacTACE-like is co-transfected with 1.0 μg of acommercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBacTACE-like are mixed in a sterile well of a microtiter platecontaining 50 μl of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μl Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27 C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27 C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg, Md.) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, Md., pages 9-10). After appropriate incubation, bluestained plaques are picked with the tip of a micropipettor (e.g.,Eppendorf). The agar containing the recombinant viruses is thenresuspended in a microcentrifuge tube containing 200 μl of Grace'smedium and the suspension containing the recombinant baculovirus is usedto infect Sf9 cells seeded in 35 mm dishes. Four days later thesupernatants of these culture dishes are harvested and then they arestored at 4 C. The recombinant virus is called V-TACE-like.

To verify the expression of the TACE-like gene, Sf9 cells are grown inGrace's medium supplemented with 10% heat inactivated FBS. The cells areinfected with the recombinant baculovirus V-TACE-like at a multiplicityof infection (“MOI”) of about 2. Six hours later the medium is removedand is replaced with SF900 II medium minus methionine and cysteine(available from Life Technologies Inc., Rockville, Md.). If radiolabeledproteins are desired, 42 hours later, 5 μCi of ³⁵S-methionine and 5 μCi³⁵S-cysteine (available from Amersham) are added. The cells are furtherincubated for 16 hours and then they are harvested by centrifugation.The proteins in the supernatant as well as the intracellular proteinsare analyzed by SDS-PAGE followed by autoradiography (if radiolabeled).Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe mature protein and thus the cleavage point and length of thesecretory signal peptide.

Example 2b Cloning and Expression of Matrilysin-Like Protein in aBaculovirus Expression System

In this illustrative example, the plasmid shuttle vector pA2 is used toinsert the cloned DNA encoding the complete protein, including itsnaturally associated secretary signal (leader) sequence, into abaculovirus to express the mature matrilysin-like protein, usingstandard methods as described in Summers et al., A Manual of Methods forBaculovirus Vectors and Insect Cell Culture Procedures, TexasAgricultural Experimental Station Bulletin No. 1555 (1987). Thisexpression vector contains the strong polyhedrin promoter of theAutographa californica nuclear polyhedrosis virus (AcMNPV) followed byconvenient restriction sites such as BamHI and Asp718. Thepolyadenylation site of the simian virus 40 (“SV40”) is used forefficient polyadenylation. For easy selection of recombinant virus, theplasmid contains the beta-galactosidase gene from E. coli under controlof a weak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate viable virus thatexpress the cloned polynucleotide.

Many other baculovirus vectors could be used in place of the vectorabove, such as pAc373, pVL941 and pAcIM1, as one skilled in the artwould readily appreciate, as long as the construct providesappropriately located signals for transcription, translation, secretionand the like, including a signal peptide and an in-frame AUG asrequired. Such vectors are described, for instance, in Luckow et al.,Virology 170:31-39 (1989).

The cDNA sequence encoding the full length matrilysin-like protein inthe deposited clone, including the AUG initiation codon and thenaturally associated leader sequence shown in FIGS. 4A-B (SEQ ID NO:4),is amplified using PCR oligonucleotide primers corresponding to the 5′and 3′ sequences of the gene.

The 5′ primer has the sequence:

5′CGCGGATCCGCCATCATGCAGCTCGTCATCTTA 3′ (SEQ ID NO:16) containing theunderlined BamHI restriction enzyme site, an efficient signal forinitiation of translation in eukaryotic cells, as described by Kozak,M., J. Mol. Biol. 196:947-950 (1987), followed by 18 bases (i.e., 46-63)of the sequence of the complete matrilysin-like protein shown in FIGS.4A-B beginning with the AUG initiation codon.

The 3′ primer (full length) has the sequence:

5′ CGCGGTACCCTCTAGTGCTTTCAGTTC 3′ (SEQ ID NO: 17) containing theunderlined Asp718 restriction site followed by 18 nucleotides (i.e.,889-906) complementary to the 3′ noncoding sequence in FIGS. 4A-B.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with BamHI and Asp718 and againis purified on a 1% agarose gel. This fragment is designated herein“F1”.

The plasmid is digested with the restriction enzymes BamHI and Asp718and optionally, can be dephosphorylated using calf intestinalphosphatase, using routine procedures known in the art. The DNA is thenisolated from a 1% agarose gel using a commercially available kit(“Geneclean” BIO 101 Inc., La Jolla, Calif.). This vector DNA isdesignated herein “V1”.

Fragment F1 and the dephosphorylated plasmid V1 are ligated togetherwith T4 DNA ligase. E. coli HB101 or other suitable E. coli hosts suchas XL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria are identified that contain the plasmid with the humanmatrilysin-like gene using the PCR method, in which one of the primersthat is used to amplify the gene and the second primer is from wellwithin the vector so that only those bacterial colonies containing theMatrilysin-like gene fragment will show amplification of the DNA. Thesequence of the cloned fragment is confirmed by DNA sequencing. Thisplasmid is designated herein pBacmatrilysin-like.

Five μg of the plasmid pBacmatrilysin-like is co-transfected with 1.0 μgof a commercially available linearized baculovirus DNA (“BaculoGold™baculovirus DNA”, Pharmingen, San Diego, Calif.), using the lipofectionmethod described by Felgner et al., Proc. Natl. Acad. Sci. USA84:7413-7417 (1987). 1 μg of BaculoGold™ virus DNA and 5 μg of theplasmid pBacmatrilysin-like are mixed in a sterile well of a microtiterplate containing 50 μl of serum-free Grace's medium (Life TechnologiesInc., Gaithersburg, Md.). Afterwards, 10 μl Lipofectin plus 90 μlGrace's medium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is rocked back and forth tomix the newly added solution. The plate is then incubated for 5 hours at27 C. After 5 hours the transfection solution is removed from the plateand 1 ml of Grace's insect medium supplemented with 10% fetal calf serumis added. The plate is put back into an incubator and cultivation iscontinued at 27 C. for four days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg, Md.) is used to alloweasy identification and isolation of gal-expressing clones, whichproduce blue-stained plaques. (A detailed description of a “plaqueassay” of this type can also be found in the user's guide for insectcell culture and baculovirology distributed by Life Technologies Inc.,Gaithersburg, Md., pages 9-10). After appropriate incubation, bluestained plaques are picked with the tip of a micropipettor (e.g.,Eppendorf). The agar containing the recombinant viruses is thenresuspended in a microcentrifuge tube containing 200 μl of Grace'smedium and the suspension containing the recombinant baculovirus is usedto infect Sf9 cells seeded in 35 mm dishes. Four days later thesupernatants of these culture dishes are harvested and then they arestored at 4 C. The recombinant virus is called V-matrilysin-like.

To verify the expression of the matrilysin-like gene, Sf9 cells aregrown in Grace's medium supplemented with 10% heat inactivated FBS. Thecells are infected with the recombinant baculovirus V-matrilysin-like ata multiplicity of infection (“MOI”) of about 2. Six hours later themedium is removed and is replaced with SF900 II medium minus methionineand cysteine (available from Life Technologies, Inc., Rockville, Md.).If radiolabeled proteins are desired, 42 hours later, 5 μCi of³⁵S-methionine and 5 μCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then they areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled). Microsequencing of the amino acidsequence of the amino terminus of purified protein may be used todetermine the amino terminal sequence of the mature protein and thus thecleavage point and length of the secretory signal peptide.

Example 3 Cloning and Expression of TACE-Like and Matrilysin-LikeProteins in Mammalian Cells

A typical mammalian expression vector contains the promoter element,which mediates the initiation of transcription of mRNA, the proteincoding sequence, and signals required for the termination oftranscription and polyadenylation of the transcript. Additional elementsinclude enhancers, Kozak sequences, and intervening sequences flanked bydonor and acceptor sites for RNA splicing. Highly efficienttranscription can be achieved with the early and late promoters fromSV40, the long terminal repeats (LTRS) from Retroviruses, e.g., RSV,HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV).However, cellular elements can also be used (e.g., the human actinpromoter). Suitable expression vectors for use in practicing the presentinvention include, for example, vectors such as PSVL and PMSG(Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be usedinclude, human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells andChinese hamster ovary (CHO) cells.

Alternatively, the gene can be expressed in stable cell lines thatcontain the gene integrated into a chromosome. The co-transfection witha selectable marker such as dhfr, gpt, neomycin, or hygromycin allowsthe identification and isolation of the transfected cells.

The transfected gene can also be amplified to express large amounts ofthe encoded protein. The DHFR (dihydrofolate reductase) marker is usefulto develop cell lines that carry several hundred or even severalthousand copies of the gene of interest. Another useful selection markeris the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175(1992)). Using these markers, the mammalian cells are grown in selectivemedium and the cells with the highest resistance are selected. Thesecell lines contain the amplified gene(s) integrated into a chromosome.Chinese hamster ovary (CHO) and NSO cells are often used for theproduction of proteins.

The expression vectors pC1 and pC4 contain the strong promoter (LTR) ofthe Rous Sarcoma Virus (Cullen et al., Molec. Cell. Biol. 5:438-447(1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985)). Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning ofthe gene of interest. The vectors contain in addition the 3′ intron, thepolyadenylation and termination signal of the rat preproinsulin gene.

Example 3(a) Cloning and Expression of TACE-Like Protein in COS Cells

The expression plasmid, pTACE-like HA, is made by cloning a cDNAencoding TACE-like into the expression vector pcDNAI/Amp or pcDNAIII(which can be obtained from Invitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767-778 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

A DNA fragment encoding the TACE-like protein is cloned into thepolylinker region of the vector so that recombinant protein expressionis directed by the CMV promoter. The plasmid construction strategy is asfollows. The TACE-like cDNA of the deposited clone is amplified usingprimers that contain convenient restriction sites, much as describedabove for construction of vectors for expression of TACE-like protein inE. coli. Suitable primers include the following, which are used in thisexample.

The 5′ primer, containing the underlined BamHI site, a Kozak sequence,an AUG start codon and codons of the 5′ coding region of the completeTACE-like protein has the following sequence:

5′ CGCGGATCCGCCATCATGTTCCTTCTCCTCGC 3′(SEQ ID NO:14).

The 3′ primer, containing the underlined XbaI site, a stop codon, and 16bp (i.e., 580-595) of 3′ coding sequence has the following sequence (atthe 3′ end):

5′CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTAACTACA CAGGCAATGT 3′ (SEQ IDNO:18).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith BamHI and XbaI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the TACE-like-encoding fragment.

For expression of recombinant TACE-like protein, COS cells aretransfected with an expression vector, as described above, usingDEAE-DEXTRAN, as described, for instance, in Sambrook et al., MolecularCloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. (1989). Cells are incubated under conditions for expressionof TACE-like protein by the vector.

Expression of the TACE-like-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample, Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and lysed with detergent-containingRIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH7.5, as described by Wilson et al. cited above. Proteins areprecipitated from the cell lysate and from the culture media using anHA-specific monoclonal antibody. The precipitated proteins then areanalyzed by SDS-PAGE and autoradiography. An expression product of theexpected size is seen in the cell lysate, which is not seen in negativecontrols.

Example 3(b) Cloning and Expression of TACE-Like Protein in CHO Cells

The vector pC4 is used for the expression of TACE-like protein. PlasmidpC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146).The plasmid contains the mouse DHFR gene under control of the SV40 earlypromoter. Chinese hamster ovary- or other cells lacking dihydrofolateactivity that are transfected with these plasmids can be selected bygrowing the cells in a selective medium (alpha minus MEM, LifeTechnologies) supplemented with the chemotherapeutic agent methotrexate.The amplification of the DHFR genes in cells resistant to methotrexate(MTX) has been well documented (see, e.g., Alt, F. W., et al., J. Biol.Chem. 253:1357-1370 (1978); Hamlin, J. L. and Ma, C., Biochem. etBiophys. Acta 1097:107-143 (1990); Page, M. J. and Sydenham, M. A.,Biotechnology 9:64-68 (1991). Cells grown in increasing concentrationsof MTX develop resistance to the drug by overproducing the targetenzyme, DHFR, as a result of amplification of the DHFR gene. If a secondgene is linked to the DHFR gene, it is usually co-amplified andover-expressed. It is known in the art that this approach may be used todevelop cell lines carrying more than 1,000 copies of the amplifiedgene(s). Subsequently, when the methotrexate is withdrawn, cell linesare obtained which contain the amplified gene integrated into one ormore chromosome(s) of the host cell.

Plasmid pC4 contains for expressing the gene of interest the strongpromoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus(Cullen et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragmentisolated from the enhancer of the immediate early gene of humancytomegalovirus (CMV) (Boshart et al., Cell 41:521-530 (1985)).Downstream of the promoter are BamHI, XbaI, and Asp718 restrictionenzyme cleavage sites that allow integration of the genes. Behind thesecloning sites the plasmid contains the 3′ intron and polyadenylationsite of the rat preproinsulin gene. Other high efficiency promoters canalso be used for the expression, e.g., the human-actin promoter, theSV40 early or late promoters or the long terminal repeats from otherretroviruses, e.g., HIV and HTLVI. Clontech's Tet-Off and Tet-On geneexpression systems and similar systems can be used to express theTACE-like protein in a regulated way in mammalian cells (Gossen, M., &Bujard, H. 1992, Proc. Natl. Acad. Sci. USA 89: 5547-5551). For thepolyadenylation of the mRNA other signals, e.g., from the human growthhormone or globin genes can be used as well. Stable cell lines carryinga gene of interest integrated into the chromosomes can also be selectedupon co-transfection with a selectable marker such as gpt, G418 orhygromycin. It is advantageous to use more than one selectable marker inthe beginning, e.g., G418 plus methotrexate.

The plasmid pC4 is digested with the restriction enzymes BamHI andAsp718 and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

The DNA sequence encoding the complete TACE-like protein including itsleader sequence is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene.

The 5′ primer has the sequence 5′CGCGGATCCGCCATCATGTTCCTTCTCCTCGC 3′(SEQ ID NO:14) containing the underlined BamHI restriction enzyme site,an efficient signal for initiation of translation in eukaryotic cells,as described by Kozak, M., J. Mol. Biol. 196:947-950 (1987), followed by17 bases (i.e., 22-38) of the sequence of the complete TACE-like proteinshown in FIG. 1, beginning with the AUG initiation codon.

The 3′ primer (full length) has the sequence:

5′ CGCGGTACCACTACACAGGCAATGT 3′ (SEQ ID NO:15) containing theunderlined, Asp7l8 restriction site followed by16 nucleotides (i.e.,580-595) complementary to the 3′ noncoding sequence in FIG. 1.

The amplified fragment is digested with the endonucleases BamHI andAsp718 and then purified again on a 1% agarose gel. The isolatedfragment and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli HB101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. 5 μg of the expression plasmid pC4 is cotransfected with0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al., supra).The plasmid pSV2neo contains a dominant selectable marker, the neo genefrom Tn5 encoding an enzyme that confers resistance to a group ofantibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 μM, 20 μM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

Example 3(c) Cloning and Expression of Matrilysin-Like Protein in COSCells

The expression plasmid, pmatrilysin-like HA, is made by cloning a cDNAencoding matrilysin-like into the expression vector pcDNAI/Amp orpcDNAIII (which can be obtained from Invitrogen, Inc.).

The expression vector pcDNAI/amp contains: (1) an E. coli origin ofreplication effective for propagation in E. coli and other prokaryoticcells; (2) an ampicillin resistance gene for selection ofplasmid-containing prokaryotic cells; (3) an SV40 origin of replicationfor propagation in eukaryotic cells; (4) a CMV promoter, a polylinker,an SV40 intron; (5) several codons encoding a hemagglutinin fragment(i.e., an “HA” tag to facilitate purification) followed by a terminationcodon and polyadenylation signal arranged so that a cDNA can beconveniently placed under expression control of the CMV promoter andoperably linked to the SV40 intron and the polyadenylation signal bymeans of restriction sites in the polylinker. The HA tag corresponds toan epitope derived from the influenza hemagglutinin protein described byWilson et al., Cell 37:767-778 (1984). The fusion of the HA tag to thetarget protein allows easy detection and recovery of the recombinantprotein with an antibody that recognizes the HA epitope. pcDNAIIIcontains, in addition, the selectable neomycin marker.

A DNA fragment encoding the matrilysin-like protein is cloned into thepolylinker region of the vector so that recombinant protein expressionis directed by the CMV promoter. The plasmid construction strategy is asfollows. The matrilysin-like cDNA of the deposited clone is amplifiedusing primers that contain convenient restriction sites, much asdescribed above for construction of vectors for expression ofmatrilysin-like protein in E. coli. Suitable primers include thefollowing, which are used in this example.

The 5′ primer, containing the underlined BamHI site, a Kozak sequence,an AUG start codon and codons of the 5′ coding region of the completematrilysin-like protein has the following sequence:

5′ CGCGGATCCGCCATCATGCAGCTCGTCATCTTA 3′ (SEQ ID NO:16).

The 3′primer, containing the underlined XbaI site, a stop codon, and 18bp (i.e., 889-906) of 3′ coding sequence has the following sequence (atthe 3′ end):

5′CGCTCTAGATCAAGCGTAGTCTGGGACGTCGTATGGGTACTCTAG TGCTTTCAGTTC 3′ (SEQ IDNO:19).

The PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digestedwith BamHI and XbaI and then ligated. The ligation mixture istransformed into E. coli strain SURE (available from Stratagene CloningSystems, 11099 North Torrey Pines Road, La Jolla, Calif. 92037), and thetransformed culture is plated on ampicillin media plates which then areincubated to allow growth of ampicillin resistant colonies. Plasmid DNAis isolated from resistant colonies and examined by restriction analysisor other means for the presence of the matrilysin-like-encodingfragment.

For expression of recombinant matrilysin-like protein, COS cells aretransfected with an expression vector, as described above, usingDEAE-DEXTRAN, as described, for instance, in Sambrook et al., MolecularCloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold SpringHarbor, N.Y. (1989). Cells are incubated under conditions for expressionof matrilysin-like protein by the vector.

Expression of the matrilysin-like-HA fusion protein is detected byradiolabeling and immunoprecipitation, using methods described in, forexample, Harlow et al., Antibodies: A Laboratory Manual, 2nd Ed.; ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1988). To thisend, two days after transfection, the cells are labeled by incubation inmedia containing ³⁵S-cysteine for 8 hours. The cells and the media arecollected, and the cells are washed and lysed with detergent-containingRIPA buffer: 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% DOC, 50 mM TRIS, pH7.5, as described by Wilson et al. cited above. Proteins areprecipitated from the cell lysate and from the culture media using anHA-specific monoclonal antibody. The precipitated proteins then areanalyzed by SDS-PAGE and autoradiography. An expression product of theexpected size is seen in the cell lysate, which is not seen in negativecontrols.

Example 3(d) Cloning and Expression of Matrilysin-Like Protein in CHOCells

The vector pC4 is used for the expression of matrilysin-like protein.Plasmid pC4 is described in detail above, in Example 3(b), whichdescribes expression of TACE-like protein in CHO cells.

The plasmid pC4 is digested with the restriction enzymes BamHI andAsp718 and then dephosphorylated using calf intestinal phosphatase byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

The DNA sequence encoding the complete matrilysin-like protein includingits leader sequence is amplified using PCR oligonucleotide primerscorresponding to the 5′ and 3′ sequences of the gene.

The 5′ primer has the sequence 5′CGCGGATCCGCCATCATGCAGCTCGTCATCTTA 3′(SEQ ID NO:16) containing the underlined BamHI restriction enzyme site,an efficient signal for initiation of translation in eukaryotic cells,as described by Kozak, M., J. Mol. Biol. 196:947-950(1987), followed by18 bases (i.e.,46-63) of the sequence of the complete matrilysin-likeprotein shown in FIGS. 4A-B, beginning with the AUG initiation codon.

The 3′ primer (full length) has the sequence:

5′ CGCGGTACCCTCTAGTGCTTTCAGTTC 3′ (SEQ ID NO: 17) containing theunderlined, Asp718 restriction site followed by 18 nucleotides (i.e.,889-906) complementary to the 3′ noncoding sequence in FIGS. 4A-B.

The amplified fragment is digested with the endonucleases BamHI andAsp718 and then purified again on a 1% agarose gel. The isolatedfragment and the dephosphorylated vector are then ligated with T4 DNAligase. E. coli HB 101 or XL-1 Blue cells are then transformed andbacteria are identified that contain the fragment inserted into plasmidpC4 using, for instance, restriction enzyme analysis.

Chinese hamster ovary cells lacking an active DHFR gene are used fortransfection. 5 μg of the expression plasmid pC4 is cotransfected with0.5 μg of the plasmid pSV2-neo using lipofectin (Felgner et al., supra).The plasmid pSV2neo contains a dominant selectable marker, the neo genefrom Tn5 encoding an enzyme that confers resistance to a group ofantibiotics including G418. The cells are seeded in alpha minus MEMsupplemented with 1 mg/ml G418. After 2 days, the cells are trypsinizedand seeded in hybridoma cloning plates (Greiner, Germany) in alpha minusMEM supplemented with 10, 25, or 50 ng/ml of metothrexate plus 1 mg/mlG418. After about 10-14 days single clones are trypsinized and thenseeded in 6-well petri dishes or 10 ml flasks using differentconcentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM).Clones growing at the highest concentrations of methotrexate are thentransferred to new 6-well plates containing even higher concentrationsof methotrexate (1 μM, 2 μM, 5 μM, 10 μM, 20 μM). The same procedure isrepeated until clones are obtained which grow at a concentration of100-200 μM. Expression of the desired gene product is analyzed, forinstance, by SDS-PAGE and Western blot or by reverse phase HPLCanalysis.

Example 4a Tissue Distribution of TACE-Like mRNA Expression

Northern blot analysis is carried out to examine TACE-like geneexpression in human tissues, using methods described by, among others,Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the TACE-like protein (SEQ ID NO: 1) is labeledwith ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labeling, theprobe is purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for TACE-like mRNA.

Multiple Tissue Northern (MTM) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with the labeled probe using ExpressHyb™ hybridizationsolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70 C. overnight, and films developed according tostandard procedures.

Example 4(b) Tissue Distribution of Matrilysin-Like Protein mRNAExpression

Northern blot analysis is carried out to examine matrilysin-like geneexpression in human tissues, using methods described by, among others,Sambrook et al., cited above. A cDNA probe containing the entirenucleotide sequence of the matrilysin-like protein (SEQ ID NO:4) islabeled with ³²P using the rediprime™ DNA labeling system (Amersham LifeScience), according to manufacturer's instructions. After labeling, theprobe is purified using a CHROMA SPIN-100™ column (ClontechLaboratories, Inc.), according to manufacturer's protocol numberPT1200-1. The purified labeled probe is then used to examine varioushuman tissues for matrilysin-like mRNA.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) are obtained from Clontech andare examined with the labeled probe using ExpressHyb™ hybridizationsolution (Clontech) according to manufacturer's protocol numberPT1190-1. Following hybridization and washing, the blots are mounted andexposed to film at −70 C overnight, and films developed according tostandard procedures.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.

Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of all publications (including patents, patentapplications, journal articles, laboratory manuals, books, or otherdocuments) cited herein are hereby incorporated by reference.

1. An isolated antibody or portion thereof that specifically binds to aprotein selected from the group consisting of: (a) a protein consistingof amino acid residues 1 to 261 of SEQ ID NO:5; (b) a protein consistingof amino acid residues 2 to 261 of SEQ ID NO:5; (c) a protein consistingof amino acid residues 23 to 261 of SEQ ID NO:5; (d) a proteinconsisting of an epitope bearing fragment of the amino acid sequence ofSEQ ID NO:5; (e) a protein consisting of a fragment of SEQ ID NO:5,wherein said fragment comprises at least 15 contiguous amino acidresidues of SEQ ID NO:5; (f) a protein consisting of a fragment of SEQID NO:5, wherein said fragment comprises at least 30 contiguous aminoacid residues of SEQ ID NO:5; (g) a protein consisting of amino acidresidues 24 to 71 of SEQ ID NO:5; (h) a protein consisting of amino acidresidues 81 to 125 of SEQ ID NO:5; (i) a protein consisting of aminoacid residues 141 to 167 of SEQ ID NO:5; (j) a protein consisting ofamino acid residues 178 to 202 of SEQ ID NO:5; and (k) a proteinconsisting of amino acid residues 212 to 260 of SEQ ID NO:5.
 2. Theantibody or portion thereof of claim 1 that specifically binds protein(a).
 3. The antibody or portion thereof of claim 1 that specificallybinds protein (b).
 4. The antibody or portion thereof of 1 thatspecifically binds protein (c).
 5. The antibody or portion thereof ofclaim 1 that specifically binds protein (d).
 6. The antibody or portionthereof of claim 1, wherein the protein said antibody binds is (e). 7.The antibody or portion thereof of claim 1 that specifically bindsprotein (f).
 8. The antibody or portion thereof of claim 1 thatspecifically binds protein (g).
 9. The antibody or portion thereof ofclaim 1 that specifically binds protein (h).
 10. The antibody or portionthereof of claim 1 that specifically binds protein (i).
 11. The antibodyor portion thereof of claim 1 that specifically binds protein (j). 12.The antibody or portion thereof of claim 1 that specifically bindsprotein (k).
 13. The antibody or portion thereof of claim 2 thatspecifically binds protein (b).
 14. The antibody or portion thereof ofclaim 1 wherein said protein specifically bound by said antibody orportion thereof is glycosylated.
 15. The antibody or portion thereof ofclaim 1 which is a monoclonal antibody.
 16. The antibody or portionthereof of claim 1 which is a polyclonal antibody.
 17. The antibody orportion thereof of claim 1 is selected from the group consisting of: (a)a chimeric antibody; (b) a humanized antibody; (c) a human antibody; and(d) a Fab fragment.
 18. The antibody or portion thereof of claim 1 whichis conjugated to a detectable substance.
 19. The antibody of claim 18wherein the detectable substance is selected from the group consistingof: (a) a radioisotopic label (b) an enzyme; (c) a fluorescent label;and (d) a chemiluminescent label.
 20. The antibody of claim 19 whereinthe radioisotopic label is selected from the group consisting of: ³H,¹¹¹In, ¹¹²In, ¹²¹I, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe,⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd and ^(99m)Tc.21. The antibody or portion thereof of claim 1 which is biotinylated.22. The antibody or portion thereof of claim 1 that specifically bindsto said protein in a Western Blot.
 23. The antibody or portion thereofof claim 1 that specifically binds to said protein in an ELISA.
 24. Acomposition comprising the antibody or portion thereof of claim 1 and acarrier.
 25. An isolated cell that produces the antibody of claim
 1. 26.A hybridoma that produces the antibody of claim
 1. 27. A method ofdetecting matrilysin-like protein in a biological sample comprising: (a)contacting a biological sample with the antibody or portion thereof ofclaim 1; and (b) detecting the antibody or portion thereof of claim 1bound to the matrilysin-like protein in the biological sample, whereinsaid antibody or portion thereof is directly or indirectly labeled witha detectable substance or compound.
 28. An isolated antibody or portionthereof produced by immunizing an animal with a protein selected fromthe group consisting of: (a) a protein whose sequence comprises aminoacid residues 1 to 261 of SEQ ID NO:5; (b) a protein whose sequencecomprises amino acid residues 2 to 261 of SEQ ID NO:5; (c) a proteinwhose sequence comprises amino acid residues 23 to 261 of SEQ ID NO:5;(d) a protein whose sequence comprises an epitope bearing fragment ofthe amino acid sequence of SEQ ID NO:5; (e) a protein comprising afragment of SEQ ID NO:5, wherein said fragment comprises at least 15contiguous amino acid residues of SEQ ID NO:5; (f) a protein comprisinga fragment of SEQ ID NO:5, wherein said fragment comprises at least 30contiguous amino acid residues of SEQ ID NO:5; (g) a protein comprisingamino acid residues 24 to 71 of SEQ ID NO:5; (h) a protein comprisingamino acid residues 81 to 125 of SEQ ID NO:5; (i) a protein comprisingamino acid residues 141 to 167 of SEQ ID NO:5; (j) a protein comprisingamino acid residues 178 to 202 of SEQ ID NO:5; and (k) a proteincomprising amino acid residues 212 to 260 of SEQ ID NO:5, wherein saidantibody or portion thereof specifically binds to the amino acidsequence of SEQ ID NO:5.
 29. The antibody or portion thereof of claim 28produced by immunizing an animal with protein (a).
 30. The antibody orportion thereof of claim 28 produced by immunizing an animal withprotein (b).
 31. The antibody or portion thereof of claim 28 produced byimmunizing an animal with protein (c).
 32. The antibody or portionthereof of claim 28 produced by immunizing an animal with protein (d).33. The antibody or portion thereof of claim 28 produced by immunizingan animal with protein (e).
 34. The antibody or portion thereof of claim28 produced by immunizing an animal with protein (f).
 35. The antibodyor portion thereof of claim 28 produced by immunizing an animal withprotein (g).
 36. The antibody or portion thereof of claim 28 produced byimmunizing an animal with protein (h).
 37. The antibody or portionthereof of claim 28 produced by immunizing an animal with protein (i).38. The antibody or portion thereof of claim 28 produced by immunizingan animal with protein (j).
 39. The antibody or portion thereof of claim28 produced by immunizing an animal with protein (k).
 40. The antibodyor portion thereof of claim 29 produced by immunizing an animal withprotein (b).
 41. The antibody or portion thereof of claim 28 whereinsaid protein specifically bound by said antibody or portion thereof isglycosylated.
 42. The antibody or portion thereof of claim 28 which is amonoclonal antibody.
 43. The antibody or portion thereof of claim 28which is a polyclonal antibody.
 44. The antibody or portion thereof ofclaim 28 which is selected from the group consisting of: (a) a chimericantibody; (b) a humanized antibody; (c) a human antibody; and (d) a Fabfragment.
 45. The antibody or portion thereof of claim 28 which isconjugated to a detectable substance.
 46. The antibody of claim 45wherein the detectable substance is selected from the group consistingof: (a) a radioisotopic label (b) an enzyme; (c) a fluorescent label;and (d) a chemiluminescent label.
 47. The antibody of claim 46 whereinthe radioisotopic label is selected from the group consisting of: ³H,¹¹¹In, ¹¹²In, ¹²¹I, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe,⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd and ^(99m)Tc.48. The antibody or portion thereof of claim 28 which is biotinylated.49. The antibody or portion thereof of claim 28 that specifically bindsto said protein in a Western Blot.
 50. The antibody or portion thereofof claim 28 that specifically binds to said protein in an ELISA.
 51. Acomposition comprising the antibody or portion thereof of claim 28 and acarrier.
 52. An isolated cell that produces the antibody of claim 28.53. A hybridoma that produces the antibody of claim
 28. 54. A method ofdetecting matrilysin-like protein in a biological sample comprising: (a)contacting a biological sample with the antibody or portion thereof ofclaim 28; and (b) detecting the antibody or portion thereof of claim 28bound to the matrilysin-like protein in the biological sample, whereinsaid antibody or portion thereof is directly or indirectly labeled witha detectable substance or compound.
 55. An isolated antibody or portionthereof that specifically binds to a protein selected from the groupconsisting of: (a) a protein consisting of the amino acid sequence ofthe full-length polypeptide encoded by the cDNA contained in ATCCDeposit Number 209055; (b) a protein consisting of the amino acidsequence of the mature polypeptide encoded by the cDNA contained in ATCCDeposit Number 209055; (c) a protein consisting of an epitope-bearingfragment of the polypeptide encoded by the cDNA contained in ATCCDeposit Number 209055; (d) a protein consisting of a fragment of thepolypeptide encoded by the cDNA contained in ATCC Deposit Number 209055,wherein said fragment comprises at least 15 contiguous amino acidresidues of the polypeptide encoded by the cDNA contained in ATCCDeposit Number 209055; and (e) a protein consisting of a fragment of thepolypeptide encoded by the cDNA contained in ATCC Deposit Number 209055,wherein said fragment comprises at least 30 contiguous amino acidresidues of the polypeptide encoded by the cDNA contained in ATCCDeposit Number
 209055. 56. The antibody or portion thereof of claim 55that specifically binds protein (a).
 57. The antibody or portion thereofof claim 55 that specifically binds protein (b).
 58. The antibody orportion thereof of claim 55 that specifically binds protein (c).
 59. Theantibody or portion thereof of claim 55 that specifically binds protein(d).
 60. The antibody or portion thereof of claim 55 that specificallybinds protein (e).
 61. The antibody or portion thereof of claim 55wherein said protein specifically bound by said antibody or portionthereof is glycosylated.
 62. The antibody or portion thereof of claim 55which is a monoclonal antibody.
 63. The antibody or portion thereof ofclaim 55 which is a polyclonal antibody.
 64. The antibody or portionthereof of claim 55 which is selected from the group consisting of: (a)a chimeric antibody; (b) a humanized antibody; (c) a human antibody; and(d) a Fab fragment.
 65. The antibody or portion thereof of claim 55which is conjugated to a detectable substance.
 66. The antibody of claim65 wherein the detectable substance is selected from the groupconsisting of: (a) a radioisotopic label (b) an enzyme; (c) afluorescent label; and (d) a chemiluminescent label.
 67. The antibody ofclaim 66 wherein the radioisotopic label is selected from the groupconsisting of: ³H, ¹¹¹In, ¹¹²In, ¹²¹I, ¹²⁵I, ¹³¹I, ³²P, ³⁵S, ¹⁴C, ⁵¹Cr,⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At, ²¹²Pb, ⁴⁷Sc,¹⁰⁹Pd and ^(99m)Tc.
 68. The antibody or portion thereof of claim 55which is biotinylated.
 69. The antibody or portion thereof of claim 55that specifically binds to said protein in a Western Blot.
 70. Theantibody or portion thereof of claim 55 that specifically binds to saidprotein in an ELISA.
 71. A composition comprising the antibody orportion thereof of claim 55 and a carrier.
 72. An isolated cell thatproduces the antibody of claim
 55. 73. A hybridoma that produces theantibody of claim
 55. 74. A method of detecting matrilysin-like proteinin a biological sample comprising: (a) contacting a biological samplewith the antibody or portion thereof of claim 55; and (b) detecting theantibody or portion thereof of claim 55 bound to the matrilysin-likeprotein in the biological sample, wherein said antibody or portionthereof is directly or indirectly labeled with a detectable substance orcompound.
 75. An isolated antibody or portion thereof produced byimmunizing an animal with a protein selected from the group consistingof: (a) a protein whose sequence comprises the amino acid sequence ofthe full-length polypeptide encoded by the cDNA contained in ATCCDeposit Number 209055; (b) a protein whose sequence comprises the aminoacid sequence of the mature polypeptide encoded by the cDNA contained inATCC Deposit Number 209055; (c) a protein whose sequence comprises anepitope-bearing fragment of the polypeptide encoded by the cDNAcontained in ATCC Deposit Number 209055; (d) a protein comprising afragment of the polypeptide encoded by the cDNA contained in ATCCDeposit Number 209055, wherein said fragment comprises at least 15contiguous ammo acid residues of the polypeptide encoded by the cDNAcontained in ATCC Deposit Number 209055; and (e) a protein comprising ofa fragment of the polypeptide encoded by the cDNA contained in ATCCDeposit Number 209055, wherein said fragment comprises at least 30contiguous amino acid residues of the polypeptide encoded by the cDNAcontained in ATCC Deposit Number
 209055. 76. The antibody or portionthereof of claim 75 produced by immunizing an animal with protein (a).77. The antibody or portion thereof of claim 75 produced by immunizingan animal with protein (b).
 78. The antibody or portion thereof of claim75 produced by immunizing an animal with protein (c).
 79. The antibodyor portion thereof of claim 75 produced by immunizing an animal withprotein (d).
 80. The antibody or portion thereof of claim 75 produced byimmunizing an animal with protein (e).
 81. The antibody or portionthereof of claim 75 wherein said protein specifically bound by saidantibody or portion thereof is glycosylated.
 82. The antibody or portionthereof of claim 75 which is a monoclonal antibody.
 83. The antibody orportion thereof of claim 75 which is a polyclonal antibody.
 84. Theantibody or portion thereof of claim 75 which is selected from the groupconsisting of: (a) a chimeric antibody; (b) a humanized antibody; (c) ahuman antibody; and (d) a Fab fragment.
 85. The antibody or portionthereof of claim 75 which is conjugated to a detectable substance. 86.The antibody of claim 85 wherein the detectable substance is selectedfrom the group consisting of: (a) a radioisotopic label (b) an enzyme;(c) a fluorescent label; and (d) a chemiluminescent label.
 87. Theantibody of claim 86 wherein the radioisotopic label is selected fromthe group consisting of: ³H, ¹¹¹In, ¹¹²In, ¹²¹I, ¹²⁵I, ¹³¹I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At,²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd and ^(99m)Tc.
 88. The antibody or portion thereof ofclaim 75 which is biotinylated.
 89. The antibody or portion thereof ofclaim 75 that specifically binds to said protein in a Western Blot. 90.The antibody or portion thereof of claim 75 that specifically binds tosaid protein in an ELISA.
 91. A composition comprising the antibody orportion thereof of claim 75 and a carrier.
 92. An isolated cell thatproduces the antibody of claim
 75. 93. A hybridoma that produces theantibody of claim
 75. 94. A method of detecting matrilysin-like proteinin a biological sample comprising: (a) contacting a biological samplewith the antibody or portion thereof of claim 75; and (b) detecting theantibody or portion thereof of claim 75 bound to the matrilysin-likeprotein in the biological sample. wherein said antibody or portionthereof is directly or indirectly labeled with a detectable substance orcompound.
 95. An isolated antibody or fragment thereof that specificallybinds a matrilysin-like protein purified from a cell culture, whereinsaid matrilysin-like protein is encoded by a polynucleotide encodingamino acids 1 to 261 of SEQ ID NO:5 operably associated with aregulatory sequence that controls the expression of said polynucleotide.96. The antibody or portion thereof of claim 95 wherein said proteinspecifically bound by said antibody or portion thereof is glycosylated.97. The antibody or fragment thereof of claim 95, wherein the antibodyor portion thereof is a monoclonal antibody.
 98. The antibody orfragment thereof of claim 95, wherein the antibody or portion thereof isa polyclonal antibody.
 99. The antibody or fragment thereof of claim 95,wherein the antibody or portion thereof is selected from the groupconsisting of: (a) a chimeric antibody; (b) a humanized antibody; (c) ahuman antibody; and (d) a Fab fragment.
 100. The antibody or portionthereof of claim 95 which is conjugated to a detectable substance. 101.The antibody of claim 100 wherein the detectable substance is selectedfrom the group consisting of: (a) a radioisotopic label (b) an enzyme;(c) a fluorescent label; and (d) a chemiluminescent label.
 102. Theantibody of claim 101 wherein the radioisotopic label is selected fromthe group consisting of: ³H, ¹¹¹In, ¹¹²In, ¹²¹I, ¹²⁵I, ¹³¹I, ³²P, ³⁵S,¹⁴C, ⁵¹Cr, ⁵⁷To, ⁵⁸Co, ⁵⁹Fe, ⁷⁵Se, ¹⁵²Eu, ⁹⁰Y, ⁶⁷Cu, ²¹⁷Ci, ²¹¹At,²¹²Pb, ⁴⁷Sc, ¹⁰⁹Pd and ^(99m)Tc.
 103. The antibody or portion thereof ofclaim 95 which is biotinylated.
 104. The antibody or fragment thereof ofclaim 95 wherein said antibody or fragment thereof specifically binds tosaid protein in a Western blot.
 105. The antibody or fragment thereof ofclaim 95 wherein said antibody or fragment thereof specifically binds tosaid protein in an ELISA.